A rapid, sensitive, and robust CE-MS method has been developed for the determination of tetracycline, oxytetracycline, and chlortetracycline in milk. Field-amplified sample stacking with electromigration injection (FASS-EMI) was used for the online concentration of tetracyclines. The conditions of separation, MS detection, and stacking were systematically optimized. The optimum buffer composition was 35 mM Tris, 1.1% formic acid, 5% methanol, and 15% ACN. By using the online concentration method of field-enhanced sample stacking (FESI)-EMI stacking, the sensitivity was increased six- to seven-fold. The RSDs (n = 6) of the relative migration time of tetracyclines were 1.1-1.4% for intraday and 2.4-2.9% for interday. The RSDs (n = 6) of the relative peak area of tetracyclines were 3.2-4.6% for intraday and 4.7-6.1% for interday. The LODs (S/N = 3) were 7.14 ng/mL for tetracycline, 11.4 ng/mL for oxytetracycline, and 14.9 ng/mL for chlortetracycline. The method has been successfully used to analyze tetracyclines residues in bovine milk.