DNA barcode detection method is widely applied for species identification, which is imperative to evaluate the effect of human economic activities on the biodiversity of ecosystem. However, the wide utilization of existing detection biosensors is limited by bulky and expensive instruments, such as Raman spectroscopy and electrochemical station. Herein, a liquid–solid triboelectric nanogenerator (TENG)‐based DNA barcode detection biosensor is proposed, which consists of water flow, fluid channel, and PDMS film attached by specifically designed capture probe. Through sequentially combining capture probe, targeted DNA barcode, and signal probe with Au nanoparticles (NPs), the surface charge density of friction layer of TENG decreases under the effect of AuNPs, verified by the density functional theory (DFT) method. Consequently, the peak value of output current spike signal for targeted DNA is smaller than that for other DNA, which is the working mechanism of the present TENG‐based biosensor. Such biosensor successfully recognizes Alvinocaris muricola among different types of Alvinocarididae shrimps, and its low limit detection can reach 1×10−12 m. The present work provides a paradigm‐shift way to develop an inexpensive and accurate technique to detect DNA barcode for species identification, and paves a novel way for the application of liquid–solid TENG.