The effects of the inoculation method of Saccharomyces bayanus BV818 and non-Saccharomyces yeast Metschnikowia agaves P3-3 and the fermentation temperature on the volatile profiles of red pitaya wine were investigated in the present study. Although the growth of P3-3 was inhibited by BV818 in the mixed inoculations, simultaneous and sequential inoculations promoted the production of seven volatiles, including higher alcohols (propan-1-ol, 3-methyl-1-butanol and phenethyl alcohol), esters (ethyl decanoate and diethyl succinate), acid (2-ethylhexanoic acid), and ketone (acetoin). Sequential inoculation produced the largest total content of volatile compounds and exhibited the best in the global aroma. The red pitaya wine produced in different inoculations can be separated by its main volatile components. Furthermore, the highest total content was yielded at 25 °C for alcohols and at 21 °C for esters and acids. Within an experimental range of 17 °C to 29 °C, the contents of benzaldehyde and acetoin decreased with the increase in temperature, whereas the change in 4-ethyl-2-methoxyphenol content was the opposite. The similarly high total contents of volatiles and global aroma score were yielded via sequential inoculation at 21 °C and 25 °C. Therefore, the desired red pitaya wine can be effectively produced by modulating the inoculation method and fermentation temperature.