BACKGROUND Protein‒protein interactions (PPIs) are the foundation of the life activities of cells. TurboID is a biotin ligase with higher catalytic efficiency than BioID or APEX that reduces the required labeling time from 18 h to 10 min. Since many proteins participate in binding and catalytic events that are very short-lived, it is theoretically possible to find relatively novel binding proteins using the TurboID technique. Cell proliferation, apoptosis, autophagy, oxidative stress and metabolic disorders underlie many diseases, and forkhead box transcription factor 1 (FOXO1) plays a key role in these physiological and pathological processes. RESULTS The FOXO1-TurboID fusion gene was transfected into U251 astrocytes, and a cell line stably expressing FOXO1 was constructed. While constructing the FOXO1 overexpression plasmid, we also added the gene sequence of TurboID to perform biotin labeling experiments in the successfully fabricated cell line to look for FOXO1 reciprocal proteins. Label-free mass spectrometry analysis was performed, and 325 interacting proteins were found. A total of 176 proteins were identified in the FOXO1 overexpression group, and 227 proteins were identified in the Lipopolysaccharide -treated group (Lipopolysaccharide, LPS). Wild-type U251 cells were used to exclude interference from nonspecific binding. The FOXO1-interacting proteins hnRNPK and RBM14 were selected for immunoprecipitation and immunofluorescence verification. CONCLUSIONS The TurboID technique was used to select the FOXO1-interacting proteins, and after removing the proteins identified in the blank group, a large number of interacting proteins were found in both positive groups. This study lays a foundation for further study of the function of FOXO1 and the regulatory network in which it is involved.