This study investigated the effect of CaO2 pretreatment on sulfonamide antibiotics (SMs) remediation by Chlorella sp. Results showed that a CaO2 dose ranging from 0.05 to 0.1 g/g biomass was the best and led to higher SMs removal efficacy 5–10% higher than the control. The contributions made by cometabolism and CaO2 in SMs remediation were very similar. Bioassimilation could remove 24% of sulfadiazine (SDZ) and sulfamethazine (SMZ), and accounted for 38% of sulfamethoxazole (SMX) remediation. Pretreatment by CaO2 wielded a positive effect on microalgae. The extracellular polymeric substances (EPS) level of the CaO2 pretreatment microalgae was three times higher when subjected to non-pretreatment. For the long-term, pretreatment microalgae removed SMs 10–20% more than the non-pretreatment microalgae. Protein fractions of EPS in continuous operation produced up to 90 mg/L for cometabolism. For bioassimilation, SMX intensity of the pretreatment samples was 160-fold less than the non-treatment one. It indicated the CaO2 pretreatment has enhanced the biochemical function of the intracellular environment of microalgae. Peroxidase enzyme involved positively in the cometabolism and degradation of SMs to several metabolites including ring cleavage, hydroxylation and pterin-related conjugation.