Keratin was extracted from chicken feather to produce scaffolds with poly(3-hydroxybutyrate) (P(3HB)) for tissue engineering applications. The scaffolds with different concentrations (10%, 20%, and 30%) of keratin were produced by electrospinning. The keratin presence in the produced scaffold was confirmed by FTIR analysis. The surface wettability of the P(3HB)/keratin scaffold was much higher compared to neat P(3HB) scaffold because of hydrophilic nature of the keratin. All the scaffolds showed over 80% porosity with an average fiber diameter of less than 750 nm. Mechanical properties of the P(3HB) scaffolds with up to 20% keratin were superior compared to neat P(3HB) scaffold. However, the mechanical properties of the P(3HB)/keratin scaffolds were deteriorated when the keratin concentration was 30% in the scaffold. The observed mechanical properties reduction was mainly attributed to the poor interaction between the keratin and P(3HB) matrix. Due to better biocompatibility of keratin, cell culture studies suggested that the P(3HB)/keratin scaffolds can have better cell attachment and proliferation compared to the neat P(3HB) scaffold. Owing to good biological response and mechanical properties, the P(3HB)/keratin scaffolds with an optimal concentration of keratin (20%) could be used as potential material for tissue engineering application.