AbstractPhage display technology is commonly applied for high‐throughput screening of single‐domain antibodies (sdAbs), and the problem of non‐specific adsorption caused by carrier proteins seriously affects the biopanning of single‐domain antibodies specific to haptens. In this paper, enrofloxacin (ENR)‐functionalized cryogels were prepared by the ethylenediamine (EDA) and carbodiimide methods for application in the biopanning of ENR‐specific phages. To improve the efficiency of biopanning, double blocking, a wash solution flow rate of 1 mL/min, and phage pre‐incubation were applied to the biopanning process through single‐factor experiments. Results of flat colony counting showed that the phage output of AG‐ENR cryogels was 15 times higher than that of AG cryogels for the same input amount. And seven complete sequences of ENR‐specific shark sdAbs were obtained by monoclonal phage ELISA and sequence alignment. All these results indicate that functionalized cryogels could be used as a novel and efficient method for phage biopanning for single‐domain antibodies to haptens.