Escherichia coli (E. coli) O157:H7 is a common foodborne pathogen which can cause serious harm. It is particularly important to establish a simple and portable method to achieve on-site pathogen detection. In this study, a capture-antibody-independent lateral flow immunoassay (LFIA) was constructed based on Cu_2−xSe nanocrystals (Cu_2−xSe NCs) for rapid detection of E. coli O157:H7. Cu_2−xSe NCs can not only be regarded as the “nano-antibody” for the recognition of E. coli O157:H7 through electrostatic adsorption, but also as nanozymes that show good peroxidase-like catalytic activity. The formed compound of E. coli O157:H7 and Cu_2−xSe NCs would be captured by a detection antibody on the T line due to the specific recognition of the antibody and E. coli O157:H7. Then, Cu_2−xSe NCs could catalyze the oxidation of TMB by H₂O₂ to generate oxTMB, thereby generating blue bands. Meanwhile, we developed a mobile app for rapid data analysis. Under the optimal reaction conditions, E. coli O157:H7 could be detected within 70 min. The detection limit of this method was 2.65 × 10⁵ CFU mL⁻¹ with good specificity and stability. Additionally, it could achieve on-site rapid detection of E. coli O157:H7 in environmental water samples, providing a promising biosensor for portable pathogen detection.