In order to heighten the biodegradation effect of the non-ionic surfactant octyl phenol ethoxylates (Octylphenyl polyethoxylates, OPnEO) in the laboratory, four mixed degradation bacteria (H1, TXBc10, OPQb11, TXBa23) were screened to degrade OPnEO. The construction of OPnEO mixed flora centers on the degradation effects of four bacterial strain of different ratios on OPnEO. The degradation rate of hybrid strain L9 (H1:TXBc10:TXBa23 1:1:1) to OPnEO reached 56.44% (highest), slightly higher than the degradation effect of each single strain. The factors related to degradation efficiency of L9 were optimized as follows:optimum carbon and nitrogen sources were sucrose and tryptone, respectively; optimum initial pH value was 7.0, the optimum temperature was 28℃; optimum inoculation amount was 4%. To further the degradation rate, three significant factors affecting the degradation OPnEO were screened in Plackett-Burman experimental design:L9 mixed bacteria inoculation, temperature, and initial pH value. Then steepest ascent approached the maximum response area of the three significant factors. Using Box-Behnken experimental design and response surface analysis, the optimal conditions for the degradation of OPnEO by mixed bacteria L9 were confirmed as follows:inoculation 4.16%, temperature 28.20℃, initial pH value 7.13, sucrose concentration 500mg/L, tryptone concentration 500mg/L, OPnEO initial concentration of 500mg/L,180r/min cultured 7d. Under these conditions, the degradation rate of OPnEO by L9 reached 62.15%, about 5% higher than non-optimized OPnEO of experimental conditions. These results can effectively solve the current problem of environmental pollution such as tanning, washing, pesticides and other industries caused by OPnEO, and will offer new ideas to the study of other types of biodegradable surfactants pollutants.