The management of chondral defects is a challenging topic of current interest for scientists and surgeons, which has a crucial impact on human cost. Even after several centuries after its first observation, this problem has still not found a satisfactory and definitive answer. Cartilage tissue engineering, which involves novel natural scaffolds, has emerged as a promising strategy for cartilage regeneration and repair. In this study, bio-plasticpoly-3-hydroxybutyrate-4-hydroxybutyrate (P34HB) film was first fabricated. The characteristics of P34HB film were tested using SEM and AFM. Cell morphologies on P34HB film were obtained using SEM and fluorescence microscopy after cell seeding. The tests of cell adhesion and proliferation on P34HB film were conducted using MTT and CCK-8 assays, respectively. Furthermore, full cartilage defects in rats were created and P34HB films were implanted to evaluate their healing effects within 8 weeks. It was found that P34HB film, as a biomaterial implant, possessed good in vitro properties for cell adhesion, migration, and proliferation. Importantly, in the in vivo experiment, P34HB film exhibited desirable healing outcomes. These results demonstrated that P34HB film was a good scaffold for cartilage tissue engineering for improving cell proliferation and adhesion.