Dunaliella salina is an important source of natural β-carotene (containing 9-cis and all trans isomers) for industrial production. The phytohormone salicylic acid (SA) has been proven to have impacts on the stress resistance of higher plants, but research on microalgae is currently unclear. In this study, the effects of SA on the growth, biochemical composition, antioxidant enzyme activity, key enzymes of β-carotene synthesis, and cis-and trans-isomers of β-carotene in D. salina under different salt concentrations were investigated. The results were shown that at concentrations of 1.5, 2, and 2.5 M NaCl, the antioxidant enzyme activity and key enzymes for β-carotene synthesis in algal cells were significantly increased, but the content and proportion of 9-cis isomer in β-carotene isomers decreased. The addition of SA significantly increased the growth and antioxidant enzyme (SOD, MDA) activity, as well as the synthesis of key enzyme phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene β cyclase (LCYB) of D. salina under high-salinity conditions. It is worth noting that under the treatment of SA, the proportion of 9-cis isomer in the three salt concentrations (1.5, 2, 2.5 M NaCl) significantly increased by 32.09%, 20.30%, and 11.32%, respectively. Moreover, SA can not only improve the salt tolerance of D. salina, but also increase the proportion of 9-cis isomer, with higher physiological activity in β-carotene, thereby enhancing the application value of D. salina.