Sepsis remains a major clinical challenge, often resulting in long-term physiological and immunological disturbances. This study employed high-throughput single-cell Raman spectroscopy to analyze the biochemical profiles of peripheral blood leukocytes from patients with non-COVID-19 and COVID-19-associated sepsis. Leukocytes were assessed at multiple timepoints, including the acute phase (Days 3 and 7 after sepsis onset) and late recovery phase (6 and 12 months after sepsis onset). Raman spectroscopic profiles of leukocytes showed clear separation between healthy controls and sepsis patients during the acute phase with high balanced accuracy (BAcc: 95%–98%). Spectral differences between acute and recovery phases (BAcc: 84%–97%) and between recovery-phase leukocytes and those from healthy controls (BAcc: 81%–90%) were also observed, indicating long-lasting molecular alterations. Furthermore, distinct profiles were identified between non-COVID-19 and COVID-19-associated sepsis during the acute phase (BAcc: 65%–71%) and in the late-recovery phase (BAcc: 71%–83%). These findings demonstrate that Raman spectroscopy enables label-free, high-throughput profiling of leukocyte biochemistry across the sepsis trajectory. This suggests that Raman spectroscopy is a promising tool for high-throughput screening, offering insights into the biomolecular changes in sepsis and providing a diagnostic platform to differentiate between sepsis etiologies, a significant advancement in the field of sepsis diagnostics.