Ginkgo biloba is an economically valuable tree, as a variety of flavonoid compounds are produced by the leaves of its seedlings. Although soil salinity is a serious threat to agricultural productivity worldwide, the effect of salt stress on G. biloba seedlings remains unclear. In this study, we found that under high NaCl concentrations (200 and 300 mmol/L), seedling growth was inhibited and the water content, chlorophyll, and peroxidase (POD) enzyme activity were significantly decreased in the leaves, whereas the soluble protein and proline levels increased significantly. However, at low NaCl concentrations (50 and 100 mmol/L), the seedlings grew normally because of the regulation of catalase (CAT) and POD enzyme activities. To elucidate the molecular mechanisms behind G. biloba salt tolerance, we examined the transcriptome of G. biloba seedlings treated with 100 mmol/L NaCl. Twelve differentially expressed genes (DEGs) were found to be involved in ion osmotic potential signal transduction and amplification, including two ABA signaling genes, five CDPK/CIPK genes, and five mitogen-activated protein kinase (MAPK) signaling genes. We also found that NAC transcription factors may be involved in the salt stress response; these included positive regulators (Gb_12203, Gb_27819, Gb_37720, and Gb_41540) and negative regulators (Gb_32549, Gb_35048, and Gb_37444). Importantly, treatment with 100 mmol/L NaCl can significantly improve flavonoid and flavonol glycoside biosynthesis. Simultaneously, the expression of flavonoid biosynthesis-related genes, including PAL (Gb_10949, Gb_21115) and FLS (Gb_00285, Gb_14024, and Gb_14029), was significantly upregulated. Based on these results, we reveal that G. biloba seedlings can tolerate low-level soil salinity stress through the regulation of different kinds of genes and transcriptome factors, especially flavonoid biosynthesis, which is improved to respond to environmental stress.