Lactulose represents the most widely studied index for distinction of heat-treated milk and for evaluating the heat load to which milk was subjected. Several methods have been reported and applied to detect lactulose in milk, including enzymatic, GC and HPLC methods. The major disadvantage of these methods is that they are laborious, time consuming and less sensitive. One of the main problems in measuring lactulose in milk is the coexistence of lactose and lactulose, which have a similar retention time using HPLC. In particular, the amount of lactose is 2 orders of magnitude larger than that of lactulose. Here we present a rapid and sensitive quantification of lactulose in milk based on the utilization of supercritical fluid chromatography coupled with mass detector. Quantification of lactulose was performed by employing selected ion recording (SIR) mode. Lactulose and lactose were well resolved from the peaks with a resolution value (Rs) of 1.8. It is worth noting that lactulose peak has a shorter retention time than lactose peak, and the tail effect is lowered. Under the optimal conditions, a linear relationship between the concentrations of lactulose and peak areas was obtained in the range of 0.1 to 10.0 mg/L (r² = 0.999), with a LOD and LOQ of 0.03 mg/L and 0.1 mg/L in matrix solution, respectively. Considering the dilution factor of sample pretreatment, the LOD and LOQ could be lower than 0.75 and 2.5 mg/L in milk sample. The relative standard deviation was 3.3% for UHT milk, indicating good precision. Compared with the methods developed using HPLC, the proposed method shows some superiorities, such as shorter retention time, less solvent consumption, better peak resolution and higher sensitivity.