Erythritol is a four-carbon sugar alcohol produced by microorganisms and is used as a natural sweetener in food industry. The present study aimed to examine the impact of various surfactants, including betaine, Tween 20, Tween 80, Span 20, and Triton X-100, on the production of erythritol by Trichosporonoides oedocephalis ATCC 16958. Finally, after 120 h culture via agitation, the supplementation with 0.5 g/L betaine increased the erythritol production by 50.38%, which was best among the five surfactants. Therefore, betaine was chosen for further research. Subsequently, the activity of erythrose reductase (ER) with the addition of 0.5 g/L betaine (0.211 U/mg protein) was slightly higher than that of the culture without betaine (0.192 U/mg protein) at 48 h of culture. However, no significant changes were observed in the ER activity with betaine addition during the fermentation stages. Interestingly, further observation with scanning electron microscope indicated that the disruption of cell was found in the culture without betaine, while that of the culture with 0.5 g/L betaine addition remained intact at 120 h post-cultivation. The enhanced erythritol production with betaine addition affected the cellular morphology that could be regulated effectively by zwitterionic surfactant betaine. Finally, the scale-up fermentation in 5-L bioreactor, erythritol production with initial 0.5 g/L betaine and 30 mg/L CuSO4·5H2O addition was up to 59.34 g/L, which was 19.59% higher than the control with only 30 mg/L CuSO4·5H2O supplementation (49.62 g/L). Thus, the present study demonstrated the mechanisms underlying the betaine-affected erythritol production and provided a feasible approach for improving the erythritol production by T. oedocephalis.