Objective: To establish a simultaneous determination method for all-trans-lycopene and β-carotene by internal standard on C30-HPLC. Methods: Stationary phase, YMCTM Carotenoid column C30 (4.6 mm×250 mm, SOD-5μm, Waters); mobile phase A, CH3CN-MeOH (3:1, V/V); mobile phase B, MTBE; both A and B with the addition of 0.05% triethylamine; elution, 30% B in 0－10 min, a linear increase of mobile phase B from 30% to 90% in 10－20 min and 90% mobile phase in 20－30 min; flow rate, 1 mL/min; PDA wavelength range, 300－600 nm; monitoring wavelength, 470 nm and 450 nm; injection volume, 20μL; column temperature, room temperature. Results: Minimum detection limit of this method was 0.01μg/mL. In the range of 0.3－0.6μg internal standard, relative correction factor of internal standard to all-trans-lycopene andβ-carotene were 1.36 and 1.22, respectively. The relative standard deviations of reproducibility experiments were 0.50% and 0.70%, respectively. The recovery rates of this method were higher than 99.00%. Conclusion: The established method is suitable for the determination for all-trans-lycopene and β-carotene in tomato and its products.