The present invention provides a method for detecting mycoplasma, which can directly confirm whether a cell is infected with mycoplasma, by simultaneously amplifying DNA extracted from lysing a host cell infected with mycoplasma, and mitochondrial DNA in the cytoplasm of the host cell. According to the method of the present invention, by directly using the DNA inside the host cell, the DNA of mycoplasma bound around the nucleus of the host cell can be used as an amplification target, thereby increasing detection sensitivity. In addition, since the mitochondrial DNA inside the host cell is used as an amplification target of an internal control sample, whether the sample has been sampled in an appropriate amount can be confirmed in a convenient manner, and furthermore, by comparing the band size between the internal control sample and the mycoplasma DNA, the degree of mycoplasma infection can be quantitatively confirmed.