Transport in water is the most common method for achieving high survival rates when transporting cultured fish in China; yet, transport success relies on proper water quality and conditions. This research was designed to explore the effects of ascorbic acid and β-1,3-glucan on survival, physiological responses, and flesh quality of farmed tiger grouper (Epinephelus fuscoguttatus) during simulated transport. The transport water temperature for live tiger grouper was 15 °C, which had the highest survival rate, the lowest stress response, and metabolic rate, and this will reduce the susceptibility to diseases. It is stated that β-1,3-glucan influences the changes of cortisol content, heat shock protein 70, IL-1β, and IgM transcription levels during simulated transport. Rather than using ascorbic acid alone (the A-group), β-1,3-glucan (3.2 mg/L) in the presence of ascorbic acid (25 mg/L) can effectively reduce the increase of transport-induced serum cortisol content, heat shock protein 70, and IL-1β, but stimulated IgM. 25 mg/L ascorbic acid and 3.2 mg/L β-1,3-glucan had no obvious effect on the nutritional indexes and flavor of live tiger grouper; however, these can effectively reduce the stress response, improve the innate immune activity, and ensure a higher survival rate.