P-nitrophenol (PNP or 4-NP) has been widely used as a biorefractory raw material in chemical industry, whereas been highly concerned for its characteristics of mutagenic/carcinogenic activity and food chain bioaccumulation. In this study, an anaerobic semi-fixed bed biofilm reactor (An-SFB-BR) was constructed and used to treat PNP wastewater which discharged from chemical industries. Experimental results revealed that the An-SFB-BR was successfully cultivated with the gradually increasing of influent PNP from 0 to 540mg/L (gradually increased 10mg/L every time in stage II and 30-50mg/L for stage III), with the observation of an average removal efficiency of 98% for PNP and 80% for chemical oxygen demand (COD), also a biogas production and biogas production rate of 2.1 L/(L·d) and 0.57 m3/kg-COD, respectively. Finally, the conversion rate of P-aminophenol (PAP), the primary intermediate of PNP reached 80% after An-SFB-BR biodegradation. A relatively stable pH was maintained throughout the entire process, and insignificant VFA accumulation. The reactor exhibited a strong toxic shock resistance, and 16S rRNA sequencing results demonstrated that the dominant microbial community changed slightly with the gradually increasing of PNP concentration, which guaranteed the PNP removal efficiency.