Herein, a facile and green one-pot route was firstly developed for in situ grown Au nanoparticles on oxygen deficient molybdenum oxide nanosheets (MoOx-AuNSs) without extrinsic reductant. The change of molybdenum-oxygen bonds and transformation of valence state of Mo element from Mo4+ and Mo5+ to Mo6+ species in MoOx after the redox reaction were observed. The nanocomposites exhibited tunable surface-enhanced Raman scattering (SERS) activity by adjusting the synthesis condition to assemble optimal hot spots. Utilization of MoOx-AuNSs as novel SERS substrate, ultrasensitive SERS detection of IR-780 iodine, a molecular probe used in biomedicine, was then achieved under different environments. Finally, the molecular fingerprint analysis of cancer cells based on MoOx-AuNSs was also completed, indicating the promising potential of MoOx-based SERS probe for bio-applications.