BACKGROUND Ticks, which are obligate blood‐feeding parasites, transmit a wide range of pathogens during their hematophagic process. Certain enzymes and macromolecules play a crucial role in inhibition of several tick physiological processes, including digestion and reproduction. In the present study, genes encoding type 2 cystatin were cloned and characterized from Haemaphysalis doenitzi, and the potential role of cystatin in tick control was further assessed. RESULTS Two cystatin genes, HDcyst‐1 and HDcyst‐2, were successfully cloned from the tick H. doenitzi. Their open reading frames are 390 and 426 base pairs, and the number of coding amino acids are 129 and 141, respectively. In the midgut, salivary glands, Malpighian tubules and ovaries of ticks, the relative expression of HDcyst‐1 was higher in the midgut and Malpighian tubules, and HDcyst‐2 was higher in the salivary glands of H. doenitzi, respectively. Lipopolysaccharide (LPS) injection and low‐temperature stress elevated cystatin expression in ticks. Enzyme‐linked immunosorbent assay showed that both rHDcyst‐1 and rHDcyst‐2 protein vaccines increased antibody levels in immunized rabbits. A vaccination trial in rabbits infected with H. doenitzi showed that both recombinant cystatin proteins significantly reduced tick engorgement weights and egg mass weight, in particular, rHDcyst‐1 significantly prolonged tick engorgement time by 1 day and reduced egg hatching rates by 16.9%. In total, rHDcyst‐1 and rHDcyst‐2 protein vaccinations provided 64.1% and 51.8% protection to adult female ticks, respectively. CONCLUSION This is the first report on the immunological characterization of the cystatin protein and sequencing of the cystatin gene in H. doenitzi. Cystatin proteins are promising antigens that have the potential to be used as vaccines for infestation of H. doenitzi control. © 2024 Society of Chemical Industry. Cystatins play a crucial role in the stress response to low‐temperature and toxin stress, and two cystatins were selected for recombinant expression to prepare protein vaccines, both of which enhanced tick control.