Abstract: A large number of organic pollutants caused serious pollution to the water cycle, and drug-resistant bacteria posed a serious threat to human health. Photocatalytic nanomaterials have become a research hotspot in the field of antibacteria. In this paper, oxygen-doped carbon nitride (O-CN) was prepared by hot polymerization with urea and formic acid. The optimal proportion of O-CN was mixed with polyvinyl alcohol (PVA) solution, and the PVA/O-CN composite nanofiber film was successfully prepared by electrospinning technology. The micro-morphology, structure, photocatalytic bacteriostatic properties and organic pollutant removal properties of O-CN and PVA/O-CN composite films were studied. The results show that O atom can replace the N position in the triazine ring structure of CN, and the light absorption capacity and the separation rate of electron-hole improve compared with that of CN. PVA/O-CN-0.6 composite nanofiber membrane has the best inhibition effect on E. coli and S. aureus, and the inhibition rates are 96% and 93.7%, respectively. In addition, PVA/O-CN-0.6 composite nanofiber membrane has a good performance in dye removal, and the removal rate of methylene blue (MB) by PVA/O-CN-0.6 reaches 97.7% within 4 h. Moreover, the film with excellent mechanical properties, has great application potential in many fields.