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In a soilless culture (perlite substrate), root restriction cannot only reduce production costs but also improve fruit quality. Therefore, this study used different levels of root restriction [T1: 0.5 L, T2: 4 L, nonrestriction treatment (CK): 35 L] on tomatoes to explore their impact on quality. Results showed that total soluble solids (TSS), glucose, fructose, and sucrose contents were increased, whereas L-tryptophan, L-tyrosine, and titratable acidity were decreased under two restriction treatments. Meanwhile, root restriction also promoted the accumulation of phenylalanine and proline. For lycopene and flavonoid biosynthesis (prunin, naringin, naringenin), the restriction groups were significantly higher than those in the control group. Overall, T1 and T2 treatment had a better effect than CK treatment. This study provided an idea for improving substrate use efficiency and tomato quality.