Kanamycin is an aminoglycoside antibiotic widely used in treating animal diseases caused by Gram-negative and Gram-positive infections. Kanamycin has a relatively narrow therapeutic index, and can accumulate in the human body through the food chain. The abuse of kanamycin can have serious side-effects. Therefore, it was necessary to develop a sensitive and selective analysis method to detect kanamycin residue in food to ensure public health. There are many analytical methods to determine kanamycin concentration, among which high performance liquid chromatography (HPLC) is a common and practical tool. This paper presents a review of the application of HPLC analysis of kanamycin in different sample matrices. The different detectors coupled with HPLC, including Ultraviolet (UV)/Fluorescence, Evaporative Light Scattering Detector (ELSD)/Pulsed Electrochemical Detection (PED), and Mass Spectrometry, are discussed. Meanwhile, the strengths and weaknesses of each method are compared. The pre-treatment methods of food samples, including protein precipitation, liquid-liquid extraction (LLE), and solid-phase extraction (SPE) are also summarized in this paper.