Methods were investigated for biofilm regeneration on carriers in a moving bed biofilm reactor used for vitamin C production wastewater treatment. Three ordinary chemical cleaning agents (hydrochloric acid, sodium hydroxide, and sodium hypochlorite) and physical drying were chosen for evaluation as methods for biofilm detachment. The results showed that these methods all had some degree of biofilm removal effectiveness. Treatment with 3% hydrochloric acid (w/w) achieved the maximum degree of biofilm detachment, at 75.2%. Biofilm biomass re-formed on carriers, from the maximum degree of biofilm detachment, quickly by an increase of 76.17 g m−2 month−1. It was concluded that treatment with 3% acid was the best choice for biofilm removal and regeneration.