Microbial polyhydroxyalkanoates (PHA) were proposed for the first time as a new type of biofuel. In this paper, poly-R-3-hydroxybutyrate (PHB) and medium chain length PHA (mcl PHA) were, respectively, esterified to become R-3-hydroxybutyrate methyl ester (3HBME) and medium chain length hydroxyalkanoate methyl ester (3HAME) via acid-catalyzed hydrolysis. The recovery percentages of 3HBME and 3HAME were 52 and 65%, respectively. The purities of 3HBME and 3HAME were 97 and 96%, respectively. Combustion heats of 3HBME, 3HAME, ethanol, n-propanol, n-butanol, 0(#) diesel, 90(#) gasoline, and 3HBME-based and 3HAME-based blended fuels were investigated and compared, respectively. It was found that 3HBME and 3HAME had combustion heats valuing 20 and 30 KJ/g, respectively. Ethanol has a combustion heat of 27 KJ/g, while addition of 10% 3HBME or 3HAME enhanced the combustion heat of ethanol to 30 and 35 KJ/g, respectively. The addition of 3HBME or 3HAME into n-propanol and n-butanol led to a slight reduction of their combustion heats. Combustion heats of blended fuels 3HBME/diesel or 3HBME/gasoline and of 3HAME/diesel or 3HAME/gasoline were lower than that of the pure diesel or gasoline. It was roughly estimated that the production cost of PHA-based biofuels should be around US$1200 per ton.