ObjectiveTo construct a Split-TurboID system based on a fragment complementation strategy to achieve efficient and specific labeling of intercellular interaction proteins.MethodsNonactive fragments of secretion-targeted Split-TurboID and direct interaction-mediated Split-TurboID were engineered, and their cellular expression and localization were validated. The labeling efficiency of the Split-TurboID system was evaluated by Western blotting, immunofluorescence and flow cytometry.ResultsThe Split-TurboID system, reconstituted through spontaneous interaction or bait protein induction, was successfully established. Western bloting, immunofluorescence, and flow cytometry analyses demonstrated that N-TurboID and C-TurboID fragments could exhibit high mutual affinity upon proximity, forming an active TurboID enzyme. Subsequent biotin labeling enabled the detection of secretory protein interactions and direct cell-cell contacts.ConclusionThe Split-TurboID technology provides an efficient, flexible, and highly specific biotinylation-based method for investigating intercellular interactions, offering a powerful tool to investigate intercellular communication.