Bioorthogonal chemistry reaction is a kind of chemical reaction that can occur under physiological conditions, which has been widely used in biomedical research field owing to its high efficiency, specificity and simplicity. Metabolic engineering based on metabolic biosynthesis pathway is a reliable modification technique, achieving the non-destructive and efficient labeling of living molecules. The various chemical reporter groups could be effectively introduced into target biomolecules via biological metabolic process, which allowing the targets were labeled with complementary probes through bioorthogonal reaction for molecular labeling and drug delivery in living systems. Combined the advantages of metabolic engineering and bioorthogonal chemistry, this labeling strategy has great application potential and research value in labeling, trace imaging and diagnosis in the fields of biomedical engineering. Herein we introduced the theory and research progress of bioorthogonal chemistry and metabolic engineering in biomedical fields, and also specially analyzed the application of bioorthogonal chemistry in molecular imaging and drug delivery.