A fragment of ribosomal protein L24 was obtained from the complementary deoxyribonucleic acid (cDNA) library of Haemaphysalis longicornis eggs. The complete sequence of the clone was subsequently obtained using rapid amplification of the cDNA ends (RACE). Ribosomal protein L24 from H. longicornis had a high percentage similarity to this protein from different species. Conserved domains were also identified in RpL24. Real-time polymerase chain reaction (PCR) analysis showed that this gene is expressed in various tissues and different developmental stages of H. longicornis. Furthermore, HLL24 is mostly expressed in ovaries and salivary glands compared with other tissues in partially fed adult female ticks, and the expression level of HLL24 is significantly lower in eggs and larvas than in other developmental stages. RpL24 was also cloned from Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum ticks, respectively. Comparison of their amino acid sequences revealed difference only in several amino acids. A vaccine based on the HLL24 recombinant protein could not protect rabbits against H. longicornis.