Nitrogen (N) sources, the critical medium component, were optimized for squalene production by microalga Aurantiochytrium sp. in heterotrophic cultures. In screening experiments monosodium glutamate, yeast extract and tryptone were found to enhance cell growth and squalene production. The optimal levels of the three nitrogen sources were further determined through central composite experimental design. The squalene content and yield were both influenced not only by monosodium glutamate, tryptone and yeast extract, but also by their interactions. The squalene content and squalene yield were described by the second-order polynomial equations with high confidence levels (>99%). The optimal concentrations of monosodium glutamate, yeast extract and tryptone were predicted to be 6.61. g/L, 6.13. g/L and 4.50. g/L for squalene content and 6.94. g/L, 6.22. g/L and 4.40. g/L for squalene yield, respectively. In the verification experiment, the squalene content and squalene yield reached 0.72. mg/g and 5.90. mg/L, respectively, which were much higher than those obtained in previous studies.