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The pomegranate is one of the most essential crop fruits in the world. 5-Aminolevulinic acid (ALA) regulates the growth and development of 5-year-old pomegranate. However, for plenty of pome crops, conventional information on the physio-chemical characteristics underlying 5-ALA is limited. Therefore, in this study, we applied four different concentrations of 5-ALA treatments (0 mg L⁻¹, 10 mg L⁻¹, 20 mg L⁻¹, 50 mg L⁻¹, and 100 mg L⁻¹), where 0 mg L⁻¹ was considered as a control group, to evaluate the effects of 5-ALA on the physiochemical characteristics of pomegranates. Our results showed that maximal photochemical efficiency <i>(F</i><i>_V/F</i><i>_M</i>), photosystem (<i>Φ_PSII</i>), and photochemical quenching (<i>qP</i>) increased at concentrations of 50 mg L⁻¹ and 100 mg L⁻¹ compared with controls. Anthocyanin contents were elevated by 2.27% and 1.33% at the 5-ALA concentrations of 50 mg L⁻¹ and 100 mg L⁻¹. Furthermore, qRT-PCRs of the DEGs, such as punica granatum chalcone synthase (<i>PgCHS</i>), chalcone isomerase<i> (PgCHI)</i>, flavanone 3-hydroxylase<i> (PgF3H)</i>, dihydroflavonol 4-reductase<i> (PgDFR)</i>, anthocyanidin synthase<i> (PgANS)</i>, and ultrafine-grained (<i>PgUFG)</i>, showed up-regulations, except for chalcone isomerase (<i>PgCHI</i>), after 5-ALA treatments. The fresh weight of the fruit and the weight of the grains were elevated under 50 mg L⁻¹ and 100 mg L⁻¹ concentrations, and both the fresh weight of the fruit and the grain weight were higher than controls. Total sugar (TS) increased by 8.49% and 24.99%, total soluble solids (TSS) increased by 2.02% and 6.07%, soluble proteins increased by 1.16% and 1.15%, and the pH level of juice increased by 0.12% and 0.19%, respectively. In addition, the contents of ascorbic acid, total phenols, and total flavonoids increased by 24.18%, 2.53%, and 1.29%, respectively, compared with controls. Taken together, the antioxidant activities of SOD and POD also increased by 13.33% and 11.95% at concentrations of 100 mg L⁻¹. Our results show that concentrations of 5-ALA treatments at 50 mg L⁻¹ and 100 mg L⁻¹ will be beneficial for crop cultivation.