Bio-demulsifier has not been applied in industrial scale due to its production cost. In order to reduce the production cost and improve the efficiency of demulsification, cheap materials such as oilfield sludge, workshop waste oil and edible waste oil were used in experiments as carbon sources to culture PRJ-1 demulsifying bacteria. As a result, cell concentrations of 5.4g/L, 3.7g/L and 4.2g/L were gotten, which were higher than the concentration of 2.6g/L cultivated by using paraffin. PRJ-1 demulsifying bacteria were used to prepare the demulsifying solution with a concentration of 10g/L, which was used in the demulsification experiment on Liaohe crude oil with a water cut of 80%. With 100 ml/L being added into the crude oil, the demulsification efficiency at 12h reached 91%, which was higher than that of 100mg/L chemical demulsifier. Demulsification efficiency of 96% can be achieved by using the combination of 60ml/L demulsifying bacteria and 40mg/L chemical demulsifier, furthermore, the demulsifying speed was higher and dehydrated water was clearer.