A new mono-ureado β-cyclodextrin-bond ordered mesoporous SBA-15 chiral stationary phase(UCDP) was prepared and packed into the column. With common methanol-water as mobile phase,the chiral pesticide diniconazole enantiomers was successfully separated by using UCDP. Effects of mobile phase composition and temperature on the chiral separation were investigated. The optimized chromatographic conditions for diniconazole enantiomers were as follows: mobile phase: methanol-water(40∶60),flow rate: 0.5mL/min,column temperature: 25℃,detection wavelength: 248nm.Based on the above separation conditions, an HPLC method for the rapid determination of diniconazole enantiomer residues in pear was established. The pear samples were extracted with acetonitrile and purified with amino solid extraction column, and determined on UCDP in the above conditions. The good linear relationships for two enantiomers were observed in the range of 0.5-125μg/mL.The average recoveries of the analytes in pear samples at two spiked concentration levels of 1.25μg/mL and 5.0μg/mL were in the ranges of 92.0%-94.4%for R-enantiomer, and 91.2%-92.8% for S-enantiomer, respectively. According to the signal-to-noise of 3(S/N=3),the detection limits of diniconazole enantiomers were 0.02μg/mL. The UCDP preparation method is simple, and the stationary phase with urea group is stable. The developed method is rapid, accurate and reproducible, and has a good application prospect in the determination of chiral pesticide residues in fruits and vegetables.