Background <p>Delayed fracture healing (DFH) is a common postoperative complication in fracture patients, and a validated serum marker may aid in the clinical management and improve the prognosis of fracture patients. In this study, we investigated the diagnostic role and potential regulatory mechanisms of miR-654-3p in DFH.</p> Methods <p>73 patients with DFH and 75 patients with normal fracture healing (NFH) were included. Expression of miR-654-3p and EMP1 and several mRNA markers of osteogenic differentiation were evaluated by RT-qPCR. The diagnostic value of miR-654-3p and EMP1 alone and in combination was assessed using ROC curves. Cell proliferation capacity was assessed by CCK-8 and apoptosis rate by flow cytometry. DLR experiments demonstrated the targeting relationship between miR-654-3p and EMP1.</p> Results <p>Levels of miR-654-3p were found to be significantly lower in DFH compared to NFH. Following cell differentiation treatment, miR-654-3p levels increased and EMP1 levels decreased. Furthermore, a negative correlation was identified between miR-654-3p and EMP1 target binding and expression levels. The combination of miR-654-3p and EMP1 holds significant diagnostic value for DFH. miR-654-3p high expression can inhibit EMP1 levels, which promotes cell proliferation, increases osteoblast activity and levels of differentiation markers, and decreases the rate of apoptosis.</p> Conclusion <p>miR-654-3p and EMP1 are aberrantly expressed in DFH, and both have high diagnostic value for DFH. miR-654-3p is involved in the proliferation, differentiation, and apoptotic activities of osteoblasts by regulating the level of EMP1, thus affecting the progression of DFH.</p>