Hydrogel-based microspheres prepared by emulsification have been widely used as drug carriers, but biocompatibility remains a challenging issue. In this study, gelatin was used as the water phase, paraffin oil was used as the oil phase, and Span 80 was used as the surfactant. Microspheres were prepared using a water-in-oil (W/O) emulsification. Diammonium phosphate (DAP) or phosphatidylcholine (PC) were further used to improve the biocompatibility of post-crosslinked gelatin microspheres. The biocompatibility of DAP-modified microspheres (0.5–10 wt.%) was better than that of PC (5 wt.%). The microspheres soaked in phosphate-buffered saline (PBS) lasted up to 26 days before fully degrading. Based on microscopic observation, the microspheres were all spherical and hollow inside. The particle size distribution ranged from 19 μm to 22 μm in diameter. The drug release analysis showed that the antibiotic gentamicin loaded on the microspheres was released in a large amount within 2 h of soaking in PBS. It was stabilized until the amount of microspheres integrated was significantly reduced after soaking for 16 days and then released again to form a two-stage drug release curve. In vitro experiments showed that DAP-modified microspheres at concentrations less than 5 wt.% had no cytotoxicity. Antibiotic-impregnated and DAP-modified microspheres had good antibacterial effects against Staphylococcus aureus and Escherichia coli, but these drug-impregnated groups hinder the biocompatibility of hydrogel microspheres. The developed drug carrier can be combined with other biomaterial matrices to form a composite for delivering drugs directly to the affected area in the future to achieve local therapeutic effects and improve the bioavailability of drugs.