In order to compound gelatin (GEL) with poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3,4HB)) to improve the hydrophilicity and biocompatibility, a simple and convenient method for preparing P(3,4HB)/GEL composite nanofibrous (P/G-CNF) was proposed. Alkyl Polyglycoside (APG) was used as an emulsifier to prepare a stable P(3,4HB)/GEL composite solution, and novel P/G-CNF was prepared by electrospinning. The intermolecular interaction, micromorphology and porosity, crystallization and mechanical properties, hydrophilicity and cytocompatibility of P/G-CNF were characterized. The results reveal that P(3,4HB), APG and GEL form many hydrogen bonds. When the m(P(3,4HB):GEL) is 10:2, the as-prepared nanofibrous exhibit the best micromorphology with a diameter and porosity of 604 nm and 68.33%. With the increasing GEL content, the crystallization and mechanical properties of P(3,4HB)/GEL composite nanofibrous scaffold (P/G-CNS) are further enhanced and decline after reaching the maximum. The hydrophilicity of P/G-CNS is significantly improved by introducing GEL. The cell proliferation rate of P/G-CNS is higher than 100%, of which the cytotoxicity grade is 0, indicating that it is non-cytotoxic. The P/G-CNS has excellent cytocompatibility and provides better cell attachment, growth and proliferation, showing promising application prospects for tissue regeneration and biomedical materials.