Most of the current graphene transistor based deoxyribonucleic acid (DNA) sensors are based on dip-and-dry methods The flow-through approach for graphene transistor based DNA sensors have not been explored yet. Moreover, the effect of probe immobilization strategies on the performance of a graphene transistor biosensor in flowing environment was rarely studied. In this paper, a microfluidic integrated reduced graphene oxide (rGO) transistor was developed for H5N1 influenza virus gene detection with high stability and sensitivity via a flow-through strategy. Different DNA probe immobilization approaches including extended long capture probe via π-π stacking, short capture probe via π-π stacking and covalent immobilization via linker were studied. Both fluorescence measurement and electrical detection were performed to evaluate the performance of rGO transistors in flowing environment for these probe immobilization strategies. The results showed that among these approaches, extended long capture probe could provide both high sensitivity and stability in flowing environment while short capture probe suffered by the low stability in flowing environment and covalent immobilization via linker had relatively low sensitivity. This microfluidic integrated rGO transistor with extended capture probe immobilization approach could provide a promising platform for nucleic acid detection with high sensitivity and stability for potential flow-through chip application.