The establishment of new insect cell lines plays important roles in the researches of insect pathology, insect toxicology, insecticide screening and activity assay, etc. Using embryos of Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) as materials, this study describes the establishment of three cell lines designated as QAU-Ho-E-3 (Ho-3), QAU-Ho-E-4 (Ho-4), and QAU-Ho-E-6 (Ho-6), respectively. Currently, the three cell lines have been passaged more than 50 times in the TNM-FH insect cell medium containing 10% fetal bovine serum (FBS). All of them showed adherent growth. The majority of Ho-3 cells are spindle-shaped, with a size of 24.35 ± 5.29 × 11.56 ± 1.67 μm. The Ho-4 cells were either spindle-shaped or oblong, with a size of 38.07 ± 8.57 × 17.62 ± 2.48 μm. The Ho-6 cells were primarily round in shape with a diameter of 14.54 ± 1.96 μm. The Ho-3 and Ho-4 cell lines contained 20 chromosomes (i.e., diploid, 2n = 20) at passages 14 and 45. The Ho-6 cell line contained 20 chromosomes (i.e., diploid, 2n = 20) at passage 14 but 40 chromosomes (i.e., polyploidy, 4n = 40) at passage 45. The results of random amplified polymorphic DNA (RAPD) analysis showed that the RAPD fingerprint of the three cell lines was consistent with that of H. oblita eggs, but clearly different from that of BTI-Tn5B1-4 and Sf-9 cells, demonstrating that the three cell lines Ho-3, Ho-4, and Ho-6 are H. oblita cell lines. The results of the growth curve test showed that the population doubling times of Ho-3, Ho-4, and Ho-6 were 101.1, 105.2, and 83.6 h, respectively. The viral infection assay indicated that these H. oblita cell lines were not permissive to infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or Bombyx mori nucleopolyhedrovirus (BmNPV).