Abstract: Nano-Se (nano-selenium) and illumination length are two important factors those are used for improving vegetable nutritional quality and yield. Although the effects of exogenous Se and photoperiod on the growth and quality of lettuce are well-studied separately, there are few studies reporting on the combination effect of these two factors. As such, in this study, we assessed the interaction of nano-Se and photoperiod on the growth and quality of lettuce; in addition, we derived the optimal combination of photoperiod and nano-Se concentration for the growth of lettuce, with the aim of providing a theoretical basis and technical starting point for plant factories to apply nano-Se under lighting systems. To accomplish this, we used ‘Italian Bolting-resistant’ lettuce (Lactuca sativa L.) in a hydroponics system with three photoperiods (light/dark: 12 h/12 h, P1; 16 h/8 h, P2; or 20 h/4 h, P3). In addition, the lettuce leaf surfaces were treated with a spray containing 0 μmol∙L⁻¹ (N1), 24 μmol∙L⁻¹ (N2), or 48 μmol∙L⁻¹ (N3) of nano-Se. The two factors were randomly combined, resulting in a total of nine photoperiod and nano-Se treatment combinations. The results revealed that first, the P2N3 treatment had a positive effect on plant height, aboveground fresh weight, underground fresh weight, and root activity. The P2N2 treatment resulted in plant height to increase significantly (P<0.05) by 13.16% and 21.74% when compared to the P1N2 and P3N2 treatments, respectively; and the P2N3 treatment resulted in the lettuce fresh weight to increase by 56.13% and 15.14% when compared to the P1N3 and P3N3 treatments, respectively, but the difference was not significant. Second, prolonging the light period increased the chlorophyll content of the lettuce, with the highest chlorophyll a and chlorophyll b contents being found in the P3N3 treatment, whereas the highest carotenoid content was found in the P3N2 treatment. Third, the soluble sugar, K, Na, and Fe contents in the lettuce increased significantly under P2 treatment, whereas the nitrate content decreased significantly (P<0.05). The P3 treatment increased the soluble protein content and reduced the sugar content. But the longer illumination period was not conducive to an accumulation of amino acids, Ca, Mg, Zn, Fe, nor Mn in the lettuce, instead, inhibitory effects were displayed. The P2N3 treatment resulted in an increased content of various amino acids in the lettuce. Interestingly, the P3 treatment in combination with nano-Se foliar spraying resulted in an increase in the amino acid content of the lettuce, however, the amino acid content decreased with an increasing nano-Se concentration. Compared with P1, the P2 and P3 treatments improved the lettuce quality, which was further improved by nano-Se leaf spraying. Finally, the principal component analysis on the growth and quality indices of the lettuce showed that 48 µmol∙L⁻¹ of nano-Se foliar spraying (i.e., the N3 treatment) under a 16 h/8 h (i.e., the P2 treatment) photoperiod resulted in the greatest improvement to lettuce growth and quality.