Abstract: Background　Cystatin C (Cys C) is a sensitive indicator for evaluating renal function. However, the clinical reference interval (RI) in use are mostly established based on adults aged 18-75 years, which are not applicable to older adults. The direct method and indirect method are two conventional methods for establishing RIs with complementary advantages. Since the indirect method has been incorporated into the industry standard in 2024, its standardized operating procedures and consistency with the gold-standard direct method require further verification. Objective　To confirm that RIs for serum cystatin C in individuals aged ≥60 years should be established independently from those aged 45 - 59 years, establish RIs using the direct method in apparently healthy elderly adults, and evaluate the applicability of indirect methods in this population. Methods　In accordance with WS/T 402-2024 industry standard, reference subjects meeting strict inclusion and exclusion criteria were prospectively enrolled from physical examination populations from March to November 2025, and the direct method was adopted to establish Cys C RIs for apparently healthy older individuals. Meanwhile, a total of 3 833 physical examination data of middle-aged and older participants were retrospectively analyzed. After screening influencing factors by multivariate linear regression analysis and removing abnormal samples, the indirect method was used to develop Cys C RIs for apparently healthy older adults in line with relevant industry standards. Z-test was performed to compare the consistency of RIs obtained by the two methods. Results　A total of 343 middle- aged reference individuals (196 males, 147 females) and 248 older reference individuals (128 males, 120 females) were enrolled in the direct method group. The results from Z-tests and nested ANOVA confirmed that the older population required separate Cys C RIs from the middle-aged people: middle-aged males, 0.75 - 1.17 mg/L; older males, 0.80 - 1.34 mg/L; middle-aged females, 0.62 - 1.14 mg/L; older females, 0.71 - 1.26 mg/L. After data cleaning and exclusion of confounding factors, 2 161 eligible middle-aged and older participants were included. In this study population, serum Cys C levels were positively correlated with age (r=0.580). Cys C levels were significantly higher in older adults than in middle-aged individuals and significantly higher in males than in females (all P＜0.01). Significant gender differences were found in both middle-aged and older groups (P＜0.01). Ultimately, 1 568 older subjects (1 356 males, 212 females) were included for indirect method analysis, with established RIs of 0.79-1.31 mg/L for older males and 0.72-1.26 mg/L for older females after Z-test grouping. The Cys C RIs derived from the two methods exhibited excellent consistency for both older males (Z=2.14＜Z*=7.46) and older females (Z=0.92＜Z*=3.53). Conclusion　Serum levels of Cys C rise with advancing age in middle-aged and older adults. It is essential to formulate age-specific and gender-stratified Cys C reference intervals for apparently healthy older adults. The indirect method with rigorous data screening shows good consistency with the direct method, and is applicable for establishing serum Cys C reference intervals in the older population.