Milk, the most consumed dairy product, is often contaminated by the carcinogenic aflatoxin M1. Here, we present a one-step automatic sample pretreatment method using immunomagnetic beads and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for the detection of aflatoxin M1 in milk with good linearity and sensitivity (detection limit = 0.0008 μg/kg and quantitation limit = 0.0027 μg/kg). Forty-five randomly purchased brands of milk from Chinese supermarkets were analyzed using the newly developed method. The positive rate of samples was 86.7%, the average content was 0.0058 μg/kg, the qualified rate was 100%, and no samples exceeded the prescribed limit (<0.025 μg/kg). The recoveries of the four different aflatoxin M1 concentrations (0.05, 0.10, 0.25, and 0.50 μg/kg) in milk samples under the optimized conditions were 86.2–102.9% with relative standard deviations (RSD) < 4.0%. The one-step automatic sample pretreatment method effectively simplifies the operation and reduces human error. Additionally, the mycotoxin automatic clean-up system used in this method can process up to 24 samples at the same time, reducing the clean-up time to ∼30 s/sample. Thus, the LC-MS/MS method offers high speed, sensitivity, and accuracy of the aflatoxin M1 quantification in milk, while being cheaper, operationally easier, and less prone to human errors than conventionally used procedures.