This study aimed to produce liquid spawn of oyster mushroom Pleurotus florida through a series of experiments to choose the suitable medium and production conditions, including the spawn storage time, fruit-body yield and biological efficiency. From experiment with 6 different liquid medium, the most suitable medium has been selected, which is GPYR (30 g glucose, 2 g peptone, 2 g yeast extract, 25 g rice bran). The suitable parameters for liquid culture were established as: pH 6, shaking speed 200 rpm, culturing time 6 days. Tune to produce liquid spawn by using 10 litre - fermentor is 4 days. The most suitable rate of spawn (dry biomass of 3.2 mg/ml) to the sawdust substrate is 3-4 percent for bags weighting from 250 to 1800 g. Storage period of liquid spawn is best within 30-45 days after production. Results from oyster mushroom cultivation with sawdust substrate showed that liquid spawn do not reduce mushroom fruit-body yield and it's biological efficiency. Moreover, by using the liquid spawn has saved 20.5 percent of spawning time and 18.0 percent of total mushroom production time.