Rapid diagnosis of aspergillosis is one of the most important aspects for disease control and treatment. The purpose of this study was to develop a metabolic fingerprint of serum from immunocompromised patients that can enable rapid detection of aspergillosis. Serum samples from immunocompromised patients with and without aspergillosis were analyzed by direct infusion mass spectrometry (DIMS). Data from the mass spectra were subjected to chemometric analysis using principal component analysis and partial least squares-discriminant analysis. Our method was able to efficiently distinguish patients with and without aspergillosis and predict the presence or absence of aspergillosis in unknown samples. Therefore, DIMS along with chemometric analysis seems to be a promising technique for rapid diagnosis of aspergillosis.