Sulfur is an essential nutrient required for plant growth and metabolism, and plays an important role in relieving stress. Nutrient deficiency is one of the main factors that negatively affect crop growth, quality, and yield. This study aimed to evaluate the effect of sulfur nutrients on the growth and metabolites of lettuce after treatment with two different sulfur concentrations (16 μM and 2 mM) in spray hydroponics. The fresh weight, chlorophyll, and carotenoid content of lettuce leaves were analyzed. Root morphology was examined using the WinRHIZO program. Metabolites were comparatively evaluated with the help of LC-MS and GC-MS. The fresh weight, chlorophyll, and carotenoid contents of lettuce were higher in the high concentration sulfur treatment group than in the low concentration sulfur treatment group. In the characteristics analysis of the lettuce roots, treatment with a high concentration of sulfur had a more positive effect on the lettuce root development than treatment with a low concentration of sulfur. Moreover, mass-based metabolomics analysis showed that the lettuce metabolites content was significantly different according to low- and high-concentration sulfur treatments. Therefore, this study highlights the importance of sulfur nutrient content in lettuce growth and metabolites.