Phage 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 is often faced during the biopanning of single-domain antibodies against haptens. Therefore, an enrofloxacin (ENR)-functionalized cryogels was prepared for application in the biopanning of ENR-specific phages, which was prepared by the ethylenediamine (EDA) and carbodiimide methods. 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. The results of flat colony counting showed that the phage output of AG-ENR cryogels was 15 times higher than the phage output of AG cryogels for the same input amount. And 7 complete sequences of ENR-specific shark sdAbs were obtained by monoclonal phage ELISA and sequence alignment. All these results indicated that AG-ENR cryogels can improve the efficiency of hapten-specific phages biopanning and this study provided a novel and efficient method for phage biopanning for single-domain antibody to haptens.