In this study, we aimed to investigate the effects of temperature and pH on the biokinetic properties (i.e., biokinetic coefficients) of thiocyanate biodegradation under a mixed-culture, autotrophic condition. Taking into account the two factors that vary simultaneously, response surface analysis (RSA), an effective statistical technique for evaluating simultaneous effects of multiple variables (Montgomery, 1997), was employed. RSA was used to design the experimental runs to test and build the response models for biokinetic coefficients. This study provides a biokinetic insight into the biodegradation process of thiocyanate.