<p>The planting of grass in controlled environmental agriculture (CEA) is useful for resolving food supply conflicts and improving land-use efficiency. However, a few studies have focused on the effects of light quality on the yield and quality of forages. Red clover (<i>Trifolium pratense</i> L.) is an important, high-protein forage worldwide and is used for treating menopausal syndrome clinically. In this study, the growth status, physiological characteristics, and secondary metabolism of <i>T. pratense</i> [cv. ‘Haifa’ (HF) and ‘Ruide’ (RD)] under red, blue, green, and white lights for 30&#xa0;days were investigated. The maximum leaf area and plant height were detected under red light. Fatty acids, including palmitic, oleic, linoleic, and arachidic acids, were also significantly increased in the stems of RD under red light (<i>P</i> &lt; 0.05). Compared with those under other lights, the stem diameter, leaf thickness, and contents of daidzein, formononetin, and biochanin A in the leaves increased under blue light. The stomatal conductance (Cond), sugar content, and <i>δ</i>¹⁵N increased under green light, but the plant height, stem diameter, and leaf area decreased. Two-way ANOVA revealed that the effects of light quality on the contents of reducing sugars, formononetin, and palmitic acid were highly significant (<i>P</i> &lt; 0.01), but the effects of cultivar were not significant (<i>P</i> &gt; 0.05). These findings illustrated that monochromatic light played a role in plant growth, photosynthesis, and secondary metabolite accumulation in <i>T. pratense</i>. Light manipulation is a promising tool for regulating the growth and quality traits of red clover and provides a rapid culture method for environmentally friendly agricultural production and breeding.</p>