Polymer blending is one of the most effective methods for providing new and desirable biocomposites for biomedical applications. To develop a new potential scaffold, biocomposite nanofibers composed of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), gelatin and laminarin were prepared using an electrospinning process. The resulting nanofibers exhibited a fully interconnected pore structure and their average diameter varied according to the mixing ratio of polymers. According to the X-ray diffraction results, the crystalline phase of PHBV was hardly affected by the addition of biopolymers. However, the crystallinity obtained from the differential scanning calorimetry data increased with a decrease of gelatin content from 30.9 to 53.3%. The water contact angles were markedly reduced by the addition of hydrophilic biopolymers from 94 degrees to 45 degrees. The results from cytocompatibility tests showed that cell proliferation on the nanofibrous scaffolds increased with increasing gelatin content, whereas the improvement of proinflammatory cytokine expression was not proportional to the gelatin content. Therefore, PHBV/gelatin/laminarin (PHGL) biocomposite nanofibrous scaffolds appear to be promising biomaterials suitable for tissue engineering.