Abstract Biomolecular piezoelectric materials show great potential in the field of wearable and implantable biomedical devices. Here, a self‐assemble approach is developed to fabricating flexible β ‐glycine piezoelectric nanofibers with interfacial polarization locked aligned crystal domains induced by Nb 2 CT x nanosheets. Acted as an effective nucleating agent, Nb 2 CT x nanosheets can induce glycine to crystallize from edges toward flat surfaces on its 2D crystal plane and form a distinctive eutectic structure within the nanoconfined space. The interfacial polarization locking formed between O atom on glycine and Nb atom on Nb 2 CT x is essential to align the β ‐glycine crystal domains with (001) crystal plane intensity extremely improved. This β ‐phase glycine/Nb 2 CT x nanofibers (Gly‐Nb 2 C‐NFs) exhibit fabulous mechanical flexibility with Young's modulus of 10 MPa, and an enhanced piezoelectric coefficient of 5.0 pC N −1 or piezoelectric voltage coefficient of 129 × 10 −3 Vm N −1 . The interface polarization locking greatly improves the thermostability of β ‐glycine before melting (≈210°C). A piezoelectric sensor based on this Gly‐Nb 2 C‐NFs is used for micro‐vibration sensing in vivo in mice and exhibits excellent sensing ability. This strategy provides an effective approach for the regular crystallization modulation for glycine crystals, opening a new avenue toward the design of piezoelectric biomolecular materials induced by 2D materials.