High content of heavy metals in the sludge was the obstacle of sludge land application and resource recycling. In this work, rhamnolipid, saponin and sophorolipid were used to enhance heavy metals removal from the sludge in the electrokinetic tests. Also, the 0.1mol L-1 HNO3 was used to control catholyte pH in the electrokinetic tests. The electric current with the duration time variation, sections pH and heavy metals residual concentrations with space and time variations were investigated in the electrokinetic tests. Results indicated that these three biosurfactants and catholyte conditioning (0.1mol L-1 HNO3) could enhance heavy metals removal from sludge in the electrokinetic experiments. After EK experiments, Cu, Zn, Cr, Pb, Ni, Mn, Fe and Hg removal efficiencies in the enhanced EK experiment were higher than unenhanced EK experiment. Cu, Zn, Cr, Pb, Ni, Mn, Fe and Hg obtained an effective removal efficiency in the EK3 test, which were 55.8 +/- 5.46%, 73.8 +/- 6.23%, 64.0 +/- 5.11%, 51.6 +/- 5.32%, 60.8 +/- 2.12%, 56.0 +/- 3.21%, 41.0 +/- 2.12%, 35.0 +/- 2.12%, respectively. Heavy metals transferring toward the cathode were greater than anode, due to the free heavy metal ions and positively charged complexed heavy metal moved to the cathode chamber by electromigration. Rhamnolipid, saponin and sophorolipid can effectively enhance heavy metals removal from the sludge in the electrokinetic tests.