Biosensors have been known as potentially powerful analytical tools for determination of the genotoxic potential of chemical compounds which are harmful to human and environment. In the present study, biosensor for detection of genotoxins was developed by design and construction of a reporter plasmid containing GFP gen under regulation of RAD54 promoter. This reporter plasmid was transformed in yeast cells which were then treated with different concentrations of 4-NQO and menadione. Due to induction of genotoxic compounds, 4-NQO at concentration of 0,5 uM triggers the activation of RAD54 promoter that regulates transcription and expresion of downstream gene, GFP, leading to fluorescence signal measured at 485 nm (excitation wave length) and 535 nm (emission wave length). Fluorescence instensity was directly proportional to 4-NQO concentrations when yeast cells treated with less than 1 uM in the environment, but decreasing when exposed to higher 4-NQO concentrations. In contrast, promoter RAD54 was not induced by menadione resulting in no fluorescence signal. Thus, such biosensor is able to identify new and uncharacterized compounds which possess the same chemical properties as 4-NQO and could be used for further development of later biosensors for new drug discovery or in drug screening.