Abstract Background Newborn screening for congenital adrenal hyperplasia (CAH) is a crucial practice with significant global adoption. While it offers substantial benefits, it also presents challenges due to high false positive rates, which can lead to mental strain for families and unnecessary hospital visits. Our research delved into the impact of birth weight (BW), gestational age (GA), and GA combined with sampling time on 17-hydroxyprogesterone (17-OHP) concentration. Specifically, we aimed to establish appropriate 17-OHP cutoff values for preterm, low birth weight weight (LBW), and sick newborns. Methods A total of 2,429 newborns born between October 2020 and January 2024 were screened for CAH. Samples were collected from neonates on a filter paper with a heel prick method. The 17-OHP concentration was measured by AutoDELFIA Neonatal 17-hydroxyprogesterone kit (PerkinElmer, Turku, Finland). This assay is a solid phase, time-resolved fluoroimmunoassay based on the competitive reaction between europium labeled 17-OHP and sample 17-OHP for a limited amount of binding sites on 17-OHP specific polyclonal antibodies. The 99.5 percentile was applied to the cutoff of 17-OHP for CAH. Results 17-OHP concentrations in newborns with lower BW and GA were significantly higher than those in normal BW and term newborns. 17-OHP concentration showed a statistically significant negative correlation with BW (r = -0.512, p < 0.001) and GA (r = -0.578, p < 0.001). Not only the full-term subgroup, but also preterm subgroups had a same tendency of 17-OHP concentration decrease with increasing sampling time. Application of newly establishing 99.5 percentile cutoff criteria significantly reduced recall rates to 1.02%, 0.81%, and 1.45% according to each criterion of BW, GA, and GA with sampling time, respectively. Conclusions This research introduces novel 17-OHP cutoff values for preterm, low birth weight (LBW), and sick newborns in the context of congenital adrenal hyperplasia (CAH). We analyzed the distribution of these cutoff values, considering factors such as birth weight, gestational age, and the timing of sample collection. Notably, applying the newly established cutoff criteria led to a significant reduction in false positive rates. Our laboratory’s data can serve as a valuable reference for other institutions aiming to set appropriate cutoffs, thereby minimizing unnecessary follow-up tests and enhancing the efficiency of CAH screening. As we continue to accumulate additional data, further reserch in this field remains promising.