Phosphorylation is a key route to achieve varieties of biological activities for polysaccharides. Here, we report the phosphorylated surface deacetylated chitin nanofibers (PS-ChNFs) using the sodium tripolyphosphate/sodium trimetaphosphate (STPP/STMP) method. Response surface methodology (RSM) was employed to optimize in this study. Under optimal conditions, a maximum degree of substitution (DS) of 0.13 was obtained. In addition, the structures of PS-ChNFs were investigated by Fourier transform infrared spectroscopy (FT-IR), Nuclear Magnetic Resonance spectra (NMR), X-ray photoelectron spectroscopy (XPS), Scanning electron microscope (SEM) and (Energy Dispersive Spectroscopy-mapping) EDS-mapping. The findings revealed that the FT-IR spectroscopy and XPS analysis confirmed the appearance of phosphate groups in PS-ChNFs. The ³¹P NMR results indicate that the PS-ChNFs structure has characteristic peaks of P elements. SEM images showed that PS-ChNFs had a rough surface with many cavities, but the P elements on the surface of the EDS-mapping are uniformly distributed throughout the sample without any enrichment. Antioxidant and antibacterial test showed that PS-ChNFs had significant scavenging effect on free radicals and antibacterial effect. The above results indicate that the chemical modification of PS-ChNFs was successful.