This experiment was carried out in 60 liters semi-continuous reactors using rice straw (RS) and water hyacinth (WH) as the substrate. All of the treatments were set up by completely random design; each treatment was done in triplicate with the loading rate at the level of 1 gVS.L-1.d-1. The results showed that mixing during incubation period enhanced 47.2 % and 6.4 % of cumulative biogas volume of WH and RS reactors, respectively. The biogas yield of WH reactors was higher than that of RS. Methane content was not significant difference from the mixing and non-mixing reactors. The maximum concentration of VFAs was 32.1, 28.6, 39.6 and 34.1 mmol/L in RSnon, RSmix, WHnon, WHmix reactors, respectively. Daily feeding was not effected to pH, the lowest pH was recorded in this experiment was 6.1 in RS reactors. RS and WH could be used as the main substrate for biogas production in semi-continuous bio-reactors.