A universal clamp sample holder was newly designed and fabricated for use with a microfluidic cotton swab-based analytical device (µCSAD) in smartphone-based colorimetric determination of sulfites in wine. In the process, sulfite (SO32−) was converted to volatile SO2 by acidification, the SO2 reduced Fe(III) to Fe(II) which finally formed the complex of Fe(II)-phenanthroline with a red color. The latter two reactions occurred on a cotton swab head hanging over the sample solution in a vial. Then, the swab was placed in the sample holder that also contained a lens and a light-emitting diode, to be photographed by the built-in camera of the smartphone. A mobile software application was designed to generate the calibration equation and evaluate the amount of sulfites in the sample. The linear ranges were 0.30–2.25 as SO2 (R2 = 0.9931) and 2.25–18.75 mg L−1 as SO2 (R2 = 0.9956). The limit of detection was 100 μg L−1. Compared to the Ripper method, the developed method showed no significant differences in measuring the sulfites in wine samples. This µCSAD is simple, cost-effectiveness, and eco-friendly. Moreover, the designed universal clamp holder and the software application can be used with multiple brands and models of smartphones.