<p>Due to the important role of vitamin D in metabolism, developing a method for rapid and high-throughput analysis of vitamin D and its metabolites is crucial. However, the current clinical diagnostic methods have the disadvantage of complicated operating procedures. In the present study, a technique combining immunocapture with mass spectrometry for rapidly detecting of 25-hydroxyvitamin D₃ in serum was developed. This method employs an antibody–antigen reaction to specifically enrich 25-hydroxyvitamin D₃ and remove complex interfering substances. Qualitative and quantitative analysis of 25-hydroxyvitamin D₃ was achieved by nano-electrospray ionization-mass spectrometry (nano-ESI–MS). The method exhibited good linearity in the range of 1–100&#xa0;ng/mL with satisfactory recovery and repeatability. The proposed method is promising for high-throughput and rapid analysis of small molecules with mass spectrometry. And it can potentially be combined with portable mass spectrometers to meet the requirements of point-of-care detection.</p>