Objective: This study aims to investigate the vitamin D nutritional status of neonates with hyperbilirubinemia and provide clinical evidence for guiding the scientific and rational supplementation of vitamin D in these neonates. Methods: This study selected neonates diagnosed with neonatal hyperbilirubinemia without other diagnoses admitted to the neonatal ward of our hospital from December 2021 to December 2023 as the research subjects. One milliliter of peripheral venous blood samples was collected from the neonates before treatment and sent to Hangzhou Qlife Precision Medical Testing Technology Co., Ltd. (Dian Diagnostics) for testing. The Sciex4500MD mass spectrometer was used as the detection instrument, and the Dicesis reagent was used as the detection reagent. The concentration of 25(OH)D in peripheral blood, namely the sum of 25(OH)D2 and 25(OH)D3 concentrations, was quantitatively detected using high-performance liquid chromatography-tandem mass spectrometry (LC-MS). According to the criteria for assessing children’s vitamin D nutritional status proposed by the Institute of Medicine of the United States in 2011 and the Global Consensus on the Management of Nutritional Rickets in 2016, serum 25-(OH)D < 30 nmol/L (12 ng/mL) was defined as vitamin D deficiency; serum 25-(OH)D 30–50 nmol/L (12– < 20 ng/mL) was defined as vitamin D insufficiency; and serum 25-(OH)D ≥ 50 nmol/L (20 ng/ml) was defined as adequate. Results: A total of 76 neonates with hyperbilirubinemia who met the diagnostic criteria were included in this study, including 40 boys and 36 girls. The results showed that the distribution of 25-(OH)D concentrations did not conform to a normal distribution, with a median of 11.69 (8.46–14.04) ng/mL. Only 5 neonates had adequate vitamin D levels, 31 had insufficient levels, and 40 had deficient levels. The rates of adequate, insufficient, and deficient vitamin D were 6.6%, 40.8%, and 52.6%, respectively, with no statistically significant difference in vitamin D levels between boys and girls (p > 0.05). Additionally, there was no statistically significant difference in serum 25(OH)D levels among neonates with hyperbilirubinemia across different seasons (F value = 0.312, p > 0.05). Conclusion: Neonates with hyperbilirubinemia generally have low vitamin D levels. However, due to the small sample size and single-center nature of this study, further verification with more samples and studies are needed.