Background: Perforin is a protein expressed in cytotoxic lymphocytes, and it plays a key role in the cytolytic effect on target cells. Mutation of the perforin gene is one of the most important pathogenic steps in primary hemophagocytic lymphohistiocytosis (pHLH). The aims of this study were to investigate the expression of perforin protein on cytotoxic lymphocytes and to determine the proportions of cytotoxic lymphocyte subsets in patients with secondary hemophagocytic lymphohistiocytosis (sHLH). Moreover, the clinical significance of these findings was explored; Methods: A total of 19 patients with sHLH and 10 healthy controls were enrolled in this study. Six of the patients achieved complete remission. Peripheral blood was collected. The protein expression of perforin on cytotoxic lymphocytes and the proportions of cytotoxic lymphocyte subsets were assessed via flow cytometry. Furthermore, clinical parameters were collected to explore correlations; Results: (1) The proportion of NK cells in the newly diagnosed sHLH was significantly lower than that in the healthy control group (p = 0.006). However, no significant difference in the proportion of CD8+ T cells or CD56+ T cells was found between the newly diagnosed sHLH group and the healthy control group (p > 0.05). (2) The expression of perforin by CD56+ T cells in the newly diagnosed sHLH group was significantly greater than that of the healthy control group (p = 0.035). However, no significant difference in the expression of perforin by CD8+ T cells or NK cells was found between the newly diagnosed sHLH group and the healthy control group (p > 0.05). (3) The proportion of CD8+ T cells in the sHLH group in complete remission was significantly lower than that in the newly diagnosed sHLH group (p = 0.023). However, no significant differences in the proportions of NK cells, CD56+ T cells, or perforin expression were found between the complete remission sHLH group and the newly diagnosed sHLH group (p > 0.05). (4) In patients with sHLH, the CD56+ T-cell proportion was positively correlated with HB (r = 0.464, p = 0.045) and negatively correlated with age (r = -0.456, p = 0.049). The proportion of NK cells was positively correlated with FIB (r = 0.531, p = 0.023). Perforin expression in CD8+ T cells was positively correlated with sCD25 (r = 0.498, p = 0.035), whereas in CD56+ T cells, it was negatively correlated with ALT (r = -0.516, p = 0.028); Conclusions: The proportion of NK cells was significantly reduced in newly diagnosed sHLH patients, whereas perforin expression in CD56+ T cells was increased. After remission, the proportion of CD8+ T cells decreased significantly. These findings suggest that monitoring cytotoxic lymphocyte subsets and perforin expression is crucial for understanding sHLH pathogenesis. The decrease in the NK cell proportion and increase in perforin expression in CD56+ T cells may reflect NK cell dysfunction and immune hyperactivation in sHLH. Additionally, CD8+ T-cell reduction after remission could contribute to clinical efficacy. Dynamic monitoring of these markers is important for evaluating treatment response.