diff --git a/tests/fuzz/oss-fuzz-build b/tests/fuzz/oss-fuzz-build
index 2fdc8c59d..bb91472f9 100755
--- a/tests/fuzz/oss-fuzz-build
+++ b/tests/fuzz/oss-fuzz-build
@@ -40,7 +40,6 @@ git submodule update --init --recursive --depth 1
         -DCMAKE_CXX_FLAGS="${CXXFLAGS}" \
         -DOT_COMPILE_WARNING_AS_ERROR=ON \
         -DOT_FUZZ_TARGETS=ON \
-        -DOT_MULTIPLE_INSTANCE=ON \
         -DOT_PLATFORM=nexus \
         -DOT_THREAD_VERSION=1.4 \
         -DOT_APP_CLI=OFF \
diff --git a/tests/nexus/build.sh b/tests/nexus/build.sh
index 4429cd8b6..b4e98e77b 100755
--- a/tests/nexus/build.sh
+++ b/tests/nexus/build.sh
@@ -64,7 +64,6 @@ echo "Building OpenThread Nexus test platform"
 echo "===================================================================================================="
 cd "${top_builddir}" || die "cd failed"
 cmake -GNinja -DOT_PLATFORM=nexus -DOT_COMPILE_WARNING_AS_ERROR=ON \
-    -DOT_MULTIPLE_INSTANCE=ON \
     -DOT_THREAD_VERSION=1.4 -DOT_APP_CLI=OFF -DOT_APP_NCP=OFF -DOT_APP_RCP=OFF \
     -DOT_15_4=${fifteenfour} -DOT_TREL=${trel} \
     -DOT_PROJECT_CONFIG="${top_srcdir}/tests/nexus/openthread-core-nexus-config.h" \
diff --git a/tests/nexus/openthread-core-nexus-config.h b/tests/nexus/openthread-core-nexus-config.h
index b3ed7aa55..6ce4969fa 100644
--- a/tests/nexus/openthread-core-nexus-config.h
+++ b/tests/nexus/openthread-core-nexus-config.h
@@ -113,6 +113,7 @@
 #define OPENTHREAD_CONFIG_MULTICAST_DNS_ENABLE 1
 #define OPENTHREAD_CONFIG_MULTICAST_DNS_ENTRY_ITERATION_API_ENABLE 1
 #define OPENTHREAD_CONFIG_MULTICAST_DNS_PUBLIC_API_ENABLE 1
+#define OPENTHREAD_CONFIG_MULTIPLE_INSTANCE_ENABLE 1
 #define OPENTHREAD_CONFIG_NAT64_BORDER_ROUTING_ENABLE 1
 #define OPENTHREAD_CONFIG_NAT64_TRANSLATOR_ENABLE 1
 #define OPENTHREAD_CONFIG_NAT64_IDLE_TIMEOUT_SECONDS 600