The study proposes a novel method of obtaining the information of second virial coefficient of interactions between microemulsion droplets by microcalorimetry. By use of a high sensitivity isothermal titration microcalorimetry to measure the dilution heat of microemulsions solution, information about the second virial coefficient of the interactions between the microemulsion droplets can be derived with the number density of microemulsion solution. The derivation is based on a hard-sphere interaction potential assumption. The variance of the derived second virial coefficients was verified by the percolation behavior of different reverse micelles solutions of dioctyl sulfosuccinate sodium salt in decane with or without solutes.