The outbreak of the disease caused by the SARS-CoV-2 virus (Covid-19) has resulted in a global health emergency that has caused millions of deaths in recent years. The control of the pandemic was significantly impacted by the availability of inputs and qualified labor to correctly diagnose the population. The challenges faced by numerous countries in conducting this extensive diagnosis, utilizing methods such as RT-PCR, emphasize the necessity for alternative testing strategies that are less reliant on expensive raw materials and can be implemented on a larger scale. This paper proposes a methodology for classifying blood serum samples as either positive or negative for Covid-19 infection using excitation-emission matrix (EEM) fluorescence spectroscopy associated with multivariate analysis. The proposed methodology uses EEM spectra of samples diagnosed by the reference method (RT-PCR) to train and validate classification models. Two approaches were tested: the first using PARAFAC and the second by unfolding the excitation-emission matrices. The DD-SIMCA model performed best in the PARAFAC approach, with an error rate of 0.05, sensitivity of 0.98 and specificity of 0.96. The PLS-DA and PCA-DA models in the second approach effectively distinguished between classes. The PCA-DA model performed the best with an error rate of 0.06 and sensitivity and specificity of 0.94. Fluorescence spectroscopy was found to be effective in analyzing serum samples and obtaining discrimination models to determine if a patient is infected with SARS-CoV-2. The findings are encouraging and could aid in the development of an inexpensive and reliable auxiliary diagnostic method.