Tropical theileriosis is a protozoan infection caused by Theileria annulata, which significantly affects cattle worldwide. This study was aimed to analyze the TaSPAG1 protein and design a novel multi-epitope vaccine candidate. Online tools were employed for the prediction of Physico-chemical properties, antigenicity, allergenicity, solubility, transmembrane domains and signal peptide, posttranslational modification (PTM) sites, secondary and tertiary structures as well as intrinsically disordered regions, followed by identification and screening of potential linear and conformational B-cell epitopes and those peptides having affinity to bind bovine major histocompatibility complex class I (MHC-I) molecules. Next, a multi-epitope vaccine construct was designed and analyzed. This 907-residue protein was hydrophilic (GRAVY: -0.399) and acidic (pI: 5.04) in nature, with high thermotolerance (aliphatic: 71.27). Also, 5 linear and 12 conformational B-cell epitopes along with 8 CTL epitopes were predicted for TaSPAG1. The 355-residue vaccine candidate had a MW of about 35 kDa and it was antigenic, non-allergenic, soluble and stable, which was successfully interacted with cattle MHC-I molecule and finally cloned into the pET28a(+) vector. Further wet studies are required to assess the vaccine efficacy in cattle. The online version contains supplementary material available at 10.1007/s40203-023-00153-5.