Abstract:[Objective] To provide theoretical basis for production and application of mix cultivated bio-demulsifier, the optimal conditions of mixed cultivation of strains L1 and XH1 were studied in this research. [Methods] Response surface methodology (RSM) based on the central composite rotation design (CCRD) was used to optimize fermentation conditions, and the demulsifying ratio was used to evaluate the demulsifying ability of fermentation broth. [Results] According to the results of the model analysis and verification, the optimal conditions were as follow: 3:2 inoculum proportion (strain L1:XH1), 3.6% (v/v) liquid paraffin contents, feeding glucose on the 4th day, and 21 h continuous incubation after feeding glucose. [Conclusion] Compared with single strain culture, the compound bio-demulsifier produced by complex culture of L1 and XH1 exhibited less dose and shorter demulsifying time. The complex culture increased the utilization ratio of main nutrients in the medium.