Objective We aimed to describe the 25-hydroxyvitamin D (25(OH)D) status of southern Chinese individuals by a high-accuracy liquid chromatography tandem mass spectrometry (LC-MS/MS) method which can trace to reference measurement procedure. Materials and methods From January 2018 to June 2019, a total of 4775 southern Chinese individuals were evaluated in our study. The serum levels of parathyroid hormone (PTH) were detected simultaneously in 162 cases. 25(OH)D was determined by LC-MS/MS, and PTH was detected using routine automated analysers. The distribution of the concentration, prevalence and seasonal variability of 25(OH)D in males and females of different age groups were studied. Results The mean 25(OH)D concentration in our study was 32.57 ng/mL (4.20-101.40 ng/mL). The global 25(OH)D concentration in males was higher than that in females of different age group. The prevalence of vitamin D deficiency (< 20 ng/mL) in females (16.65%) was higher than that in males (6.83%). The prevalence of vitamin D deficiency (< 20 ng/mL) was most common in winter (22.98% of all women and 15.49% of all men). 25(OH)D concentrations were higher in those from whom blood samples were collected in summer and autumn than in winter and spring. 25(OH)D-2 was detected in 672 serum samples (14.07%). In addition, there was a negative correlation between the concentrations of 25(OH)D and serum PTH (r = - 0.149, P < 0.05). Conclusion Our study demonstrated that the average serum 25(OH)D concentration in southern Chinese individuals was higher than that in other Chinese cohorts by a high-accuracy LC-MS/MS method. The global 25(OH)D concentration in males was higher than that in females of different ages, and the prevalence of vitamin D deficiency in females was higher than that in males. Seasonal change was an important aspect of 25(OH)D concentration in young and middle-aged people but became less relevant for that in older subjects. 25(OH)D-2 detection was of minor practical significance in our study. In addition, we also found that there was a negative correlation between the serum levels of 25(OH)D and PTH in southern Chinese individuals.