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2D Janus thin films have received increasing attentions due to their unique  asymmetric structures and fantastic applications. Herein, a macroscopic 2D freestanding Janus carbon nanotubes/gold nanoparticles (CNTs/AuNPs)  hybrid ultrathin film is successfully constructed at the air/liquid interface, using polydopamine modified unilaterally CNTs ultrathin network film as an asymmetrical 2D interfacial reactor for in situ selective growth of AuNPs on the unilateral water-side (the side facing water) of CNTs film. The resultant macroscopic 2D freestanding Janus CNTs/AuNPs ultrathin film could be used directly as electrode materials and modify stably electrode with improved  stability and operation feasibility. More importantly, the convenient and clean modification of Janus CNTs/AuNPs film on electrode is favorable to the electrons transmission in the electrode and remarkably improves the electrochemical sensitivity of glassy carbon, showing the ability of really exerting asymmetrical functionality of Janus 2D materials in a collaborative strategy.