Bowen's disease (BD) is a relatively rare early-stage squamous cell carcinoma in situ, most commonly affecting the middle-aged and elderly, and occurring on the skin or mucous membranes of various parts of the body. Its onset is concealed, the course of the disease is chronic, and some patients have malignant tumors outside the skin; therefore, it is necessary to diagnose and evaluate the disease at an early stage. This study aimed to investigate the application of reflectance confocal microscopy (RCM) in the diagnosis of BD. We performed RCM imaging on the lesion site and underwent skin biopsy for histological diagnosis of 92 patients initially diagnosed with BD in clinic. A retrospective analysis of the RCM result as well as the histological examination revealed that after analyzing RCM images, out of 92 biopsy lesions, 61 were diagnosed with BD, of which 54 were consistent with RCM diagnosis. Among the 59 cases diagnosed with BD by RCM, 54 cases were consistent with the histological diagnosis. Afterwards, we analyzed the RCM characteristics in patients with BD verified by biopsy, and compared the RCM images of two different lesions, classic Bowen's disease and pigmented Bowen's disease, and further summarized the key points of BD under RCM. Finally, we focused on the differential characteristics between BD and other skin diseases in RCM. RCM is of great value in the diagnosis of BD. RESEARCH HIGHLIGHTS: A retrospective study of RCM and histological diagnosis in patients with clinical diagnosis of BD. Analyze the RCM characteristics of skin lesions verified by biopsy. RCM is of great value in the diagnosis and differentiation of BD.