Objective: To analyze and evaluate the value of the anti-epidermal intercellular desmosome antibodies, anti-desmoglein (Dsg) 1, and anti-Dsg3, as well as the anti-epidermal basement membrane hemidesmosomes antibodies, anti-BP180, and anti-BP230 in the diagnosis of pemphigus and bullous pemphigoid. Methods: Patients with pemphigus or bullous pemphigoid treated in the Department of Dermatology of Dalian Dermatosis Hospital from July 2019 to July 2021 were selected. They were clinically diagnosed with histopathological and indirect immunofluorescence methods. They were divided into the pemphigus group (n = 102) and the bullous pemphigoid group (n = 175). Additionally, 120 patients who were ruled out of pemphigus and bullous pemphigoid during the same period were selected as the control group. Enzyme-linked immunosorbent assay (ELISA) was used to detect anti-Dsg1, anti-Dsg3, anti-BP180, and anti-BP230 antibodies. Indirect immunofluorescence (IIF) was used to detect the IgG levels of the anti-epidermal intercellular desmosome antibodies and anti-epidermal basement membrane hemidesmosomes antibodies. The positive rate, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic rate of each antibody were evaluated and analyzed comprehensively. Results: In the pemphigus group, bullous pemphigoid group, and control group, the positive rates of anti-Dsg1 are respectively 83.3% (85/102), 0 (0/175), and 1.7% (2/120); the positive rates of anti-Dsg3 are 41.1% (42/102), 0 (0/175), and 0 (0/120), respectively; the positive rates of BP180 are 5.9% (6/102), 85.7% (150/175), and 5% (6/120), respectively; the positive rates of BP230 are 1.9% (2/102), 57.7% (101/175), and 1.7% (1/120), respectively. Meanwhile, the positive rates of anti-epidermal intercellular desmosome antibodies are 69.6% (71/102), 0 (0/175), and 0 (0/120), respectively; the positive rates of anti-epidermal basement membrane hemidesmosome antibodies are 0% (0/102), 51.4% (90/175), and 0 (0/120), respectively. Among the patients with pemphigus, the sensitivity, specificity, PPV, NPV, and diagnostic rates of the anti-epidermal intercellular desmosome antibody test were 65.1%, 100%, 100%, 85.2%, and 92.2%; for anti-Dsg1, respectively, 83.3%, 99.3%, 97.7%, 88.7%, and 95.2%; for anti-Dsg3, respectively, 41.1%, 100%, 100%, 83.1%, and 84.9%. Among the patients with bullous pemphigoid, the sensitivity, specificity, PPV, NPV, and diagnostic rates of the anti-epidermal basement membrane hemidesmosomes antibody test were 51.4%, 100%, 100%, 72.3%, and 78.6%; for anti-BP180, respectively, 85.7%, 94.6%, 92.6%, 89.4%, and 90.7%; for anti-BP230, respectively, 57.7%, 98.2%, 96.2%, 74.7%, and 80.1%. Conclusion: The detection of autoantibodies in serum and the confirmation of the specific target antigens could complement each other to reduce clinical missed diagnosis and increase the positive diagnostic rate if the two tests were conducted simultaneously. The positive result of the anti-epidermal intercellular desmosome antibody and the anti-epidermal basement membrane hemidesmosomes antibody has better accuracy in diagnosing pemphigus and bullous pemphigoid. In contrast, the negative result is of great value in ruling out pemphigus and bullous pemphigoid.