Volatile fatty acids (VFAs) production process via anaerobic digestion from rice straw is highly promising due to the preferable economic value of mixed shortchain fatty acids. However, the VFA fermentation has critical disadvantage as low digestion rate of rice straw owing to the relatively short digestion time. To increase the VFAs yield and enhance rice straw utilization, the anaerobic digestion (AD) digestate from the VFAs production was exploited for the second stage VFAs production. The first stage VFAs production was conducted by three different pretreatment methods and three different fermentation temperatures, thus the AD digestate of all sets after fermentation was used as substrates for the second stage VFAs production, and VFAs potential production for all AD digestate sets still needed to be investigated. The AD digestate of all sets was directly fermented at the same temperature of 35℃ for the second stage VFAs production without further additional pretreatment. The results revealed that all sets can have more stable and suitable pH value at the second stage. Both hydrothermal and 2% HCl sets had higher VFAs yield at the second stage. The maximum VFAs yield was achieved under the condition of 1% Ca(OH)2 pretreatment and temperature of 35℃. By combining the two stages together, the total VFAs yield of 1% Ca(OH)2 group was enhanced from 0.07~0.20g/g at the first stage to 0.27~0.38g/g in total. The mass balance analysis was based on the hypotheses that the initial substrate was 100g rice straw and 1% Ca(OH)2 pretreatment was chosen at the first stage. The 1% Ca(OH)2 and 35℃ group produced 12.24g VFAs at the first stage and 11.69g VFAs at the second stage. The VFAs yield for the combined twostage was 95.5% greater than that of the first stage. The research result could provide useful reference for enhancing VFAs yield by further fermentation of the AD digestate obtained from the first stage.