Entropy is a thermodynamic property toward equilibrium based on the dissipation of energy. Cells constitute such a thermodynamic system, in which entropy production is both inevitable and highly significant. Although the experimental measurement of entropy production in a cell is very difficult, a new method to accomplish this in living cells is reported herein. Through heating the sample by alternating electric fields and recording the heat flow from cells, the entropy production in two normal cell lines, MCF10A and HL-7702, and two cancerous cell lines, MDA-MB-231 and SMMC-7721, was measured and compared. The scaled electroinduced entropy production rate (SEEP) of cancer cells monotonically increases with electric field strength at 5–40 V/cm, while that of normal cells changes nonmonotonically with electric field strength, reaching a peak at 5–30 V/cm. For all cell lines, the cancerous-to-normal ratio of field-induced entropy production is clearly <1 in a large range of field strength from 5 to 25 V/cm. Therefore, this work presents an easy and effective strategy for experimentally investigating the thermodynamic properties of the cell, and gives deeper insight into the physical differences between normal and cancerous cells exposed to electric fields.