Removal of paintings from old backings is the most critical step in the process of remounting the paintings. The paste used in the process of mounting often makes it hard to separate the painting itself from the support paper. As a consequence, the removal process is very complex and easy to result in damage of ancient paintings and calligraphy. In the traditional method of removal, the object is moistened with water for a long time in order to reduce the viscosity of the paste, and then a conservator use his/her fingers to gently twist the support paper, making possible removal of the old support paper and paste from the back of the painting. In the present study, the extracellular fluid of Bacillus fermentation was used to make a biological removal agent. The effectiveness of the agent on the aged cotton paper was tested. The results showed that the biological agent obtained by using the fermentation broth of Bacillus subtilis, after dialysis to eliminate bacteria, had good effectiveness. The safety of using a biological agent to remove paintings was tested by scanning electron microscopy and tensile strength analysis. It is found that the biological agent can reduce the peeling force and fiber index, increase the tensile strength of paper samples, compared to water moistening, and has no significant effect on the paper microstructure. The agent improves the safety of removing backing papers. Good results have been achieved on treating a complete set of Ming Dynasty land and water paintings.