diff --git a/tools/otci/otci/__init__.py b/tools/otci/otci/__init__.py
index 7cb0aa45b83b..eed56c615ada 100644
--- a/tools/otci/otci/__init__.py
+++ b/tools/otci/otci/__init__.py
@@ -37,7 +37,8 @@
     connect_ncp_sim, \
     connect_cmd_handler, \
     connect_otbr_ssh, \
-    connect_otbr_adb
+    connect_otbr_adb_tcp, \
+    connect_otbr_adb_usb
 
 from .types import Rloc16, ChildId, NetifIdentifier
 
@@ -46,7 +47,8 @@
     'connect_cli_serial',
     'connect_ncp_sim',
     'connect_otbr_ssh',
-    'connect_otbr_adb',
+    'connect_otbr_adb_tcp',
+    'connect_otbr_adb_usb',
     'connect_cmd_handler',
 ]
 
diff --git a/tools/otci/otci/command_handlers.py b/tools/otci/otci/command_handlers.py
index 7c790e6d839d..ac24e4a641b0 100644
--- a/tools/otci/otci/command_handlers.py
+++ b/tools/otci/otci/command_handlers.py
@@ -285,19 +285,13 @@ def set_line_read_callback(self, callback: Optional[Callable[[str], Any]]):
 
 class OtbrAdbCommandRunner(OTCommandHandler):
 
-    def __init__(self, host, port):
-        from adb_shell.adb_device import AdbDeviceTcp
-
-        self.__host = host
-        self.__port = port
-        self.__adb = AdbDeviceTcp(host, port, default_transport_timeout_s=9.0)
+    from adb_shell.adb_device import AdbDevice
 
+    def __init__(self, adb: AdbDevice):
+        self.__adb = adb
         self.__line_read_callback = None
         self.__adb.connect(rsa_keys=None, auth_timeout_s=0.1)
 
-    def __repr__(self):
-        return f'{self.__host}:{self.__port}'
-
     def execute_command(self, cmd: str, timeout: float) -> List[str]:
         sh_cmd = f'ot-ctl {cmd}'
 
@@ -324,3 +318,32 @@ def wait(self, duration: float) -> List[str]:
 
     def set_line_read_callback(self, callback: Optional[Callable[[str], Any]]):
         self.__line_read_callback = callback
+
+
+class OtbrAdbTcpCommandRunner(OTCommandHandler):
+
+    def __init__(self, host: str, port: int):
+        from adb_shell.adb_device import AdbDeviceTcp
+
+        self.__host = host
+        self.__port = port
+
+        adb = AdbDeviceTcp(host, port, default_transport_timeout_s=9.0)
+        super(OtbrAdbTcpCommandRunner, self).__init__(adb)
+
+    def __repr__(self):
+        return f'{self.__host}:{self.__port}'
+
+
+class OtbrAdbUsbCommandRunner(OtbrAdbCommandRunner):
+
+    def __init__(self, serial: str):
+        from adb_shell.adb_device import AdbDeviceUsb
+
+        self.__serial = serial
+
+        adb = AdbDeviceUsb(serial, port_path=None, default_transport_timeout_s=9.0)
+        super(OtbrAdbUsbCommandRunner, self).__init__(adb)
+
+    def __repr__(self):
+        return f'USB:{self.__serial}'
diff --git a/tools/otci/otci/otci.py b/tools/otci/otci/otci.py
index 46a73734db26..93bb56a67bec 100644
--- a/tools/otci/otci/otci.py
+++ b/tools/otci/otci/otci.py
@@ -33,7 +33,7 @@
 from typing import Callable, List, Collection, Union, Tuple, Optional, Dict, Pattern, Any
 
 from . import connectors
-from .command_handlers import OTCommandHandler, OtCliCommandRunner, OtbrSshCommandRunner, OtbrAdbCommandRunner
+from .command_handlers import OTCommandHandler, OtCliCommandRunner, OtbrSshCommandRunner, OtbrAdbTcpCommandRunner, OtbrAdbUsbCommandRunner
 from .connectors import Simulator
 from .errors import UnexpectedCommandOutput, ExpectLineTimeoutError, CommandError, InvalidArgumentsError
 from .types import ChildId, Rloc16, Ip6Addr, ThreadState, PartitionId, DeviceMode, RouterId, SecurityPolicy, Ip6Prefix, \
@@ -2503,8 +2503,13 @@ def connect_otbr_ssh(host: str, port: int = 22, username='pi', password='raspber
     return OTCI(cmd_handler)
 
 
-def connect_otbr_adb(host: str, port: int = 5555):
-    cmd_handler = OtbrAdbCommandRunner(host, port)
+def connect_otbr_adb_tcp(host: str, port: int = 5555):
+    cmd_handler = OtbrAdbTcpCommandRunner(host, port)
+    return OTCI(cmd_handler)
+
+
+def connect_otbr_adb_usb(serial: str):
+    cmd_handler = OtbrAdbUsbCommandRunner(serial)
     return OTCI(cmd_handler)