Tomatoes are sensitive to low temperatures. The frequent occurrence of prolonged suboptimal temperature stress in recent years has posed significant threats to tomato production. To mitigate these effects, an experiment was conducted using the tomato variety ‘Yuanshuai No. 1’ in a controlled climate chamber. Seven treatments were set up: normal temperature (day/night 25/16±1℃), which was the normal temperature control (CK); suboptimal temperature (day/night 16/8±1℃), which was the suboptimal temperature control (CKL), foliar spraying of EBR which was EBR treatment, root application of 5mL·L−1HIDS which was H1 treatment , root application of 15mL·L−1HIDS which was H2 treatment, combined application of 5mL·L−1HIDS and EBR which was E+H1 treatment, and combined application of 15mL·L−1HIDS and EBR which was E+H2 treatment. The effects of root application of HIDS and foliar spraying of EBR (brassinolide) on tomato growth, photosynthesis, yield, and quality under suboptimal temperature during the seedling stage were studied. The results showed that suboptimal temperature inhibited tomato growth, leading to shorter plants, reduced photosynthetic capacity, and lower yield and quality. Under suboptimal temperature, the EBR treatment alone increased stem diameter, improved net photosynthetic rate (Pn), and significantly increased the number of fruits by 21.48% compared to CKL. With the addition of 5mL·L−1 HIDS (H1) to the nutrient solution under suboptimal temperature, all growth indicators of tomato showed an upward trend compared to CKL, with increased photosynthetic pigment content and Pn, improved yield, and higher contents of soluble protein, soluble sugar, reduced Vc, and free amino acids. When foliar spraying of EBR was combined with H1 (E+H1), the leaf area of tomatoes increased significantly by 52.63%. With the addition of 15mL·L−1 HIDS (H2) under suboptimal temperature, there were improvements in growth indicators, photosynthetic capacity, fruit yield, and quality. When EBR was also applied (E+H2), the stem diameter increased by 19.04% compared to that affected by CKL, chlorophyll and carotenoid contents also increased, and fruit yield significantly rose by 22.23%, along with higher contents of soluble protein, soluble sugar, and reduced Vc. Thus, under suboptimal temperature during the seedling stage, both individual and combined applications of HIDS and EBR led to upward trends in tomato growth indicators, increased stem diameter, shortened internode length, increased leaf area, enhanced Pn, and improved fruit yield and quality. The combination of root application of 15mL·L−1HIDS and foliar spraying of EBR (E+H2) showed the best results.