The COVID-19 pandemic has prompted interest in identifying reliable biomarkers to predict disease severity and guide clinical decisions. Prolactin (PRL), a hormone traditionally associated with lactation, has gained attention for its role in immune modulation. This study aimed to assess PRL as a biomarker for disease severity in COVID-19. A prospective cohort of 142 patients with moderate to severe COVID-19, defined as a WHO-CPS 5 or 6, was recruited from the University General Hospital of Patras. Baseline PRL levels were measured using an electrochemiluminescence immunoassay, and serum cytokines, including IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF-α, were quantified through flow cytometry. Clinical outcomes, including mortality and the need for invasive mechanical ventilation (IMV), were recorded. Results indicated that PRL levels were significantly higher in female patients (12.95 ng/mL vs. 9.40 ng/mL, p < 0.001) but they did not correlate with key severity indices such as CCI, SOFA score upon admission or inflammatory markers. No significant associations between baseline PRL levels, cytokine concentrations, and clinical outcomes in COVID-19 were noted. Our findings suggest that PRL may lack prognostic reliability for disease severity compared to more established predictive markers and that its role in the immune response remains uncertain.