Distribution, patterns and prognostic impact of spontaneous antibody responses against different tumor-associated antigens (TAAs) in malignant melanoma patients are unknown so far and were investigated in this study for the first time in a large cohort at different stages of the disease, identifying new prognostic biomarkers for malignant melanoma. Serum samples from 365 melanoma patients (97 stage I melanoma patients, 87 stage II, 92 stage III, 89 stage IV) and 100 age and gender matched healthy control donors were analyzed. Samples were drawn at the time of diagnosis (stage I-III) or at time of diagnosis of distant metastasis (stage IV). Applying a novel multiplex assay, humoral immune responses against 29 TAAs were determined and the association between response and patient survival was investigated Antibody responses were mainly found in melanoma patients and all tested antigens elicited immune responses in all disease stages. Antibody responses against single antigens were either associated with poor prognosis and/or shorter progression-free survival (PFS) or had no influence. While in stages I-III significant associations were observed between an antibody response and overall survival or PFS, among stage IV patients, no significant association was found. Multivariate analyses identified specific humoral immune responses as prognostic factors independently of age, chemotherapy and immunotherapy. Antibody responses against specific TAA in stage I-III melanoma patients correlate with poor prognosis and/or shorter PFS. These results may help to design clinical studies in order to evaluate the potential of these responses as prognostic serological biomarkers. © 2014 Wiley Periodicals, Inc.