Abstract : Objective To explore the levels of serum soluble programmed cell death protein-1 ( sPD-1) in healthy individuals and its influence factors ，and preliminarily establish the reference intervals of the senum sPD-1 level in healthy individuals. Methods The study included 375 healthy individuals who underwent physical examination in Nanjing Drum Tower Hospital and Jurong Hospital from May 2023 to September 2023. The research objects consisted of 194 males and 181 females. The senum samples were collected and and the levels of sPD-1 in the serum samples were detected by previous est ablished fluorescence immunoassay based on new -tpe probe bio-sensor, ie., tests-on-probes ( T0Ps). The impacts of gender, age and other laboratory indicators on sPD-1 level were analyzed. The reference intervals of serum sPD-1 levels among the overall population and the individuals across various three age ranges were estimated according to CLSI C28-A3 guidelines. Results The serum sPD-1 level in healthy population was 108.82 (92.80, 134. 95)pg/ mL. There was no signifcant difference of serum sPD-1 levels between healthy males and females ( P>0.05). Two groups with age of <65 years old and≥65 years old were divided, and the serum sPD-1 level in the group over 65 years old were significantly higher than that in the group less than 65 years old [ 106.65 (90.35, 130.60) pg/mL vs 113.20 (95.60, 144.83) pg/mL, P=0.014]. When the healthy individuals were divided into three groups ( <40 years, 40 to 64 years and≥65 years), the levels of serum sPD-1 were 112.00 (98.68, 137.23 )pg/mL,9.07 ( 82.38, 1 19.60) pg/mL and 113.20 ( 95.60, 144.83) respectively and statistical sigifcances were still found among three groups ( P<0.001 ). Multiple linear regression showed the levels of GGT was a influencing factor of serum sPD-1 levels with negative correlation in healthy population. Based on the nonparametic method recommended by C28-A3 document of Clinical and Laboratory Standards Institutes ( CLSI), the reference intervals of serum sPD-1 levels in the range of the 2.5" to 97.5"percentiles were calculated as 66.7 to 214.7 pg/mL in the overall healthy population, 71.9 to 202.7 pg/mL in those <40 years old,59.4 to 220.0 pg/ mL in those between 40 and 64 years old and 69.6 to 229.0 pg/mL in those≥65 years old respectively. Conclusion This study preliminarily revealed the reference interval of serum sPD-1 level among healthy individuals as well as three various age groups.