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Magnetic resonance imaging (MRI) is limited in clinical application due to its low sensitivity. Hyperpolarization technology overcomes this limitation by enhancing molecular MRI signal. At present, ［1-13C］pyruvate is the most important hyperpolarized biological probe, which is widely used in hyperpolarized MRI research. In this study, an efficient two-step sequence of nucleophilic addition reaction and ozonation reaction for synthesis of ［1-13C］pyruvate from 13CO2 was developed, with the yield of 71.5%, and the chemical purity was above 98%. Furthermore, the ozonation reaction was optimized to furnish the one-pot synthesis of ［1-13C］pyruvate in 70.5% yield, and the chemical purity was above 98%. The synthesis of ［1-13C］pyruvate provides a basis for the development of magnetic resonance imaging research.