The invention discloses a personalized medication gene detection kit and application. The personalized medication gene detection kit is suitable for multiple kinds of sample types including dried blood spots, whole blood and buccal swabs, information of all medicine gene polymorphic sites can be acquired at once, the sequencing effective rate is 50% or above, the sequencing cover degree is 100%, the average sequencing depth exceeds 3000 X, the library building success rate is larger than or equal to 98.5%, and the medicine gene polymorphic site detection accuracy is larger than or equal to 99.9%; a primer group is high in specificity and little in nonspecific amplification, interference among primers can be well overcome, under the situation of up to 186 primer sequences, one-tube multiplePCR reaction is achieved, moreover, amplicon is high in uniformity, and under the situation of large amplification area GC content difference, overall effective amplification can be conducted. The detection kit, the primer group and a library building method can be applied to personalized medication gene detection, and guidance is provided for personalized medication.