Piezoelectric biomaterials have attracted great attention owing to the recent recognition of the impact of piezoelectricity on biological systems and their potential applications in implantable sensors, actuators, and energy harvesters. However, their practical use is hindered by the weak piezoelectric effect caused by the random polarization of biomaterials and the challenges of large-scale alignment of domains. Here, we present an active self-assembly strategy to tailor piezoelectric biomaterial thin films. The nanoconfinement-induced homogeneous nucleation overcomes the interfacial dependency and allows the electric field applied in-situ to align crystal grains across the entire film. The β-glycine films exhibit an enhanced piezoelectric strain coefficient of 11.2 pm V−1and an exceptional piezoelectric voltage coefficient of 252 × 10−3Vm N−1. Of particular significance is that the nanoconfinement effect greatly improves the thermostability before melting (192 °C). This finding offers a generally applicable strategy for constructing high-performance large-sized piezoelectric bio-organic materials for biological and medical microdevices.