The invention provides a preparation process of doxycycline hydrochloride. In the preparation process, 5-hydroxy-6-demethyl-6-desoxy-6-methylene-11-chlorotetracycline is pressurized and hydrogenated with hydrogen in the presence of catalyst-palladium/carbon (Pd/C) and adjuvant and then reacts with 5-sulfosalicylic acid to obtain alpha-6-doxycycline 5-sulfosalicylate; then alpha-6-doxycycline base is generated after basification; and finally the doxycycline hydrochloride is generated through salt transformation. The preparation process of the doxycycline hydrochloride has the advantages that the productivity and stereoselectivity are improved, the content of related substances in the finished product is reduced, and the high-quality finished product is obtained.