Objectives: This study aimed to investigate the distinguishing ability of lymphocyte subtyping for diagnosis and prognosis of invasive fungal disease (IFD). Methods: We assessed lymphocyte subtyping and evaluated the quantitative changes in key immunological parameters at intensive care unit (ICU) admission in critically ill patients at high risk and their potential influence on diagnosis and outcome of IFD. The primary outcome was 28-day mortality. Results: Among the 124 critically ill patients with mean Candida score 3.89 (0.76), 19 (15.3%) were in the IFD group. CD28⁺CD8⁺ T-cell counts (area under the curve [AUC] 0.899, 95% confidence interval [CI], 0.834-0.964, P < .001) had better distinguishing ability than other immune parameters for IFD diagnosis. The cutoff value of CD28⁺CD8⁺ T-cell counts at ICU admission for IFD diagnosis was 59.5 cells/mm³, with 83.3% sensitivity and 86.4% specificity. Multivariate logistic regression analysis identified CD28⁺CD8⁺ T-cell count <59.5 cells/mm³ (odds ratio 59.7, 95% CI, 7.33-486.9, P < .001) as an independent predictor for IFD diagnosis. CD28⁺CD8⁺ T-cell counts could also predict 28-day mortality (AUC 0.656, 95% CI, 0.525-0.788, P = .045). Kaplan-Meier survival analysis provided evidence that natural killer cell count <76.0 cells/mm³ (log-rank test; P = .001), CD8⁺ T-cell count <321.5 cells/mm³ (log-rank test; P = .04), and CD28⁺CD8⁺ T-cell count <129.0 cells/mm³ (log-rank test; P = .02) at ICU admission were associated with lower survival probabilities. Conclusion: CD28⁺CD8⁺ T-cell counts play an important role in early diagnosis of IFD. Low counts are associated with early mortality in critically ill patients at high risk of IFD. Our findings add evidence to the utility of lymphocyte subtyping in a diagnostic algorithm to better define IFD in critically ill patients at high risk.