The red-fleshed dragon fruit (Hylocereus polyrhizus) is a popular fruit in tropical countries due to its taste and nutritional value. This study aimed to investigate the physiological and biochemical changes during the development and maturation of red-fleshed dragon fruit grown in Vietnam. Fruits were measured for size and content of chlorophyll, carotenoids, reducing sugars, starch, total organic acids, vitamin C, lipids and proteins from 6 to 34 days after anthesis (DAA). The fruits reached a maximum size at 32 DAA. Chlorophyll content increased gradually from fruit formation to 18 DAA, then rapidly decreased until fruit ripening, whereas carotenoid content increased gradually from fruit formation to ripening. Starch and total organic acids contents gradually increased and reached maximum values at 18 and 22 DAA, respectively, and then declined. The contents of reducing sugars, lipids and vitamin C increased as the fruit proceeded towards ripening, reached maximum values at 32 DAA and then declined once the fruit was overripe. Proteins content gradually increased from 6 to 14 DAA and then decreased as the fruit proceeded towards ripening. These results suggest that red-fleshed dragon fruit should be harvested at 32 DAA to maximize the nutritional value and quality of the fruit.