Vitamin D deficiency has been associated with various diseases and adverse health outcomes, including diabetes, cardiovascular diseases, and cancer. Currently, the majority of vitamin D testing is conducted in large-scale laboratories upon the request of a physician as part of an annual panel of blood tests. In this paper, we present a novel paper-based microfluidic system that enables the rapid quantification of vitamin D levels on a portable platform. The system comprises a smartphone accessory, an app, and a test strip that allows for the colorimetric detection of 25-hydroxyvitamin D3 using a gold-nanoparticle-based immunoassay. We demonstrate that the system can accurately measure the physiological levels of 25-hydroxyvitamin D with an accuracy higher than 25 nM and a precision of 10 nM. We compared our system with the well-established ELISA test kits for serum samples of unknown concentration and demonstrated the equivalency of the obtained results.