diff --git a/onnxruntime/core/providers/webnn/builders/impl/conv_op_builder.cc b/onnxruntime/core/providers/webnn/builders/impl/conv_op_builder.cc
index af3293dd3d92c..b37340624f850 100644
--- a/onnxruntime/core/providers/webnn/builders/impl/conv_op_builder.cc
+++ b/onnxruntime/core/providers/webnn/builders/impl/conv_op_builder.cc
@@ -251,8 +251,18 @@ Status ConvOpBuilder::AddToModelBuilderImpl(ModelBuilder& model_builder, const N
         std::vector<int64_t> input_shape;
         ORT_RETURN_IF_NOT(GetShape(*input_defs[0], input_shape, logger), "Cannot get shape");
         for (size_t i = 0; i < 2; i++) {
-          total_padding[i] = strides[i] * (narrow<size_t>(input_shape[i + 1]) - 1) +
-                             output_padding[i] + ((kernel_shape[i] - 1) * dilations[i] + 1) - output_shape[i];
+          // Get the dimensions of H and W.
+          // For NHWC layout, the dimensions of H and W correspond to index 1 and 2.
+          // For NCHW layout, the dimensions of H and W correspond to index 2 and 3.
+          if (model_builder.GetPreferredLayout() == DataLayout::NHWC) {
+            total_padding[i] = strides[i] * (narrow<size_t>(input_shape[i + 1]) - 1) +
+                               output_padding[i] + ((kernel_shape[i] - 1) * dilations[i] + 1) - output_shape[i];
+          } else {
+            ORT_RETURN_IF_NOT(model_builder.GetPreferredLayout() == DataLayout::NCHW,
+                              "WebNN GPU backend preferred layout should be NCHW.");
+            total_padding[i] = strides[i] * (narrow<size_t>(input_shape[i + 2]) - 1) +
+                               output_padding[i] + ((kernel_shape[i] - 1) * dilations[i] + 1) - output_shape[i];
+          }
         }
         pads[0] = total_padding[0] - (total_padding[0] / 2);
         pads[1] = total_padding[0] / 2;