This study introduces a novel method of solid phase extraction (SPE), preconcentration and HPLC determination of 2-mercaptobenzimidazole (2MBI), 2-mercaptobenzoxazole (2MBO) and 2-mercaptobenzothiazole (2MBT) from an aqueous solution by a SPE cartridge loaded with copper oxide nanoparticles. Results demonstrated that copper oxide nanoparticles are quite efficient for extraction and preconcentration of trace amounts of these mercaptans at room temperature. The study also investigated the effects of parameters such as pH, buffer and its volume, electrolyte concentration, flow rate of the test solution, composition and volume of the desorbing solvent, accepted tolerable volume, amount of adsorbent, reusability of cartridges and evidence of some co-existing species on extraction and determination of the above mentioned mercaptans. The method showed good linearity for determination of these mercaptans in the range of 0.01-10 μg mL(-1) with regression coefficients better than 0.9969. The limits of detection (LODs) evaluations were 0.0021, 0.0027 and 0.0019 μg mL(-1) for 2MBT, 2MBO and 2MBI, respectively. The relative standard deviations (RSDs) for 0.2 μg mL(-1) and 5 μg mL(-1) of the measured mercaptans were below 3.04% and 4.23%, respectively. Ramin Power Plant (3000 MW, Ahvaz, Iran) cooling water containing some 2MBT (as corrosion inhibitor) was used as the real sample. Recovery tests with spiked levels of 2MBT, 2MBI and 2MBO were carried out and satisfied results were obtained.