As a promising environmental protection technology, the application of ultraviolet B irradiation in vegetable production has been widely considered. However, the effect of UVB irradiation varies with different plant varieties. In this study, we investigated the effects of two UVB intensities (0.7, 1.4 W m⁻²) on the accumulation of phenolics and flavonoids, and antioxidant activity of green-leaf and red-leaf lettuce (Lactuca sativa L.) 7 days prior to harvest. The results indicated that short-term (within 2 days) UVB treatment could promote the increase in total chlorophyll content of red-leaf lettuce and green-leaf lettuce, which increased by 49.8% and 20.6% compared with day zero, respectively, and was beneficial to the synthesis of carotenoids of red-leaf lettuce. Extending UVB exposure time significantly decreased chlorophyll a/b value of green-leaf lettuce from 0.92 to 0.63, and simultaneously increased the accumulation of antioxidant substances such as flavonoids, which were increased by 90.0% and 183.4% compared with day zero for UVB-0.7 and UVB-1.4 treatments of red-leaf lettuce, 84.1% and 110.9% of green-leaf lettuce. In contrast, red-leaf lettuce had a higher accumulation level of secondary metabolites, faster scavenging rate of free radicals, and stronger ability to resist UVB stress. Our results suggest that supplementation of low-dose UVB radiance prior to harvest can improve the secondary metabolite content and antioxidant activity of the two kinds of lettuce. This research provided a theoretical basis for improving lettuce quality by pre-harvest UVB treatment in controlled environmental agriculture.