In order to study the influence factors of the expression activities of D-Hydantoinase (D-Hase) and N-Carbamoyl hydrolase (D-Case) from Ensifer adhaerens CAS22-03, and to improve the catalytic efficiency of the whole-cell catalyzed preparation of D-p-Hydroxyphenylglycine (D-p-HPG) by Ensifer adhaerens CAS22-03, the single factor test and orthogonal test were adopted to optimize the carbon and nitrogen sources, and the fed-batch fermentation process of Ensifer adhaerens CAS22-03 was established. The enzymatic properties of D-Hase and D-Case were analyzed, and the D-p-HPG enzymatic production process based on the whole cell catalysis of Ensifer adhaerens CAS22-03 was developed. The results showed that the optimal carbon and nitrogen sources for Ensifer adhaerens CAS22-03 fermentation were sucrose and yeast extract. In the fed-batch fermentation process, the activities of D-Hase and D-Case were up to 243.6 and 55.8 U/g, which were increased by 56.9% and 46.4%, respectively. The optimal reaction temperature of D-Hase and D-Case is 45℃, and the optimal reaction pH is 9 and 8 respectively. In the process of the whole-cell catalytic preparation of D-p-HPG by Ensifer adhaerens CAS22-03, when the substrate concentration was 40 g/L and the enzyme dosage was substrate:bacterium=5:1, the substrate conversion reached more than 95% with the condition of 40℃ and 200 r/min for 10 h, laying the foundation for the industrial enzymatic production of D-p-HPG.