A method was developed for the determination of ochratoxin A (OTA) in human urine by HPLC-FLD after molecularly imprinted polymer solid phase extraction (MIP-SPE) column. After the pH being adjusted to 2.5 with 0.1 mol x L(-1) HC1, sample was cleaned up with MIP-SPE column for ochratoxin A, the analyte was analyzed by high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD), and finally all the positive results were confirmed by LC-MS/MS. Recoveries from urine samples spiked with OTA at levels ranging from 2 to 20 ng x mL(-1) were 90.6%-101.9%, and RSDs were 0.1%-1.6%. Sixty-five volunteers living in Beijing took part in the study, of which 5 were found containing OTA in their urine and the highest value was 0.091 ng x mL(-1). The MIP-SPE column was firstly applied to purify and concentrate OTA in human urine, this method is simple, rapid and reliable and can be used to determine the contents of OTA in human urine.