Objective: The elevated choline transporters (ChT), choline kinase (ChK), choline uptake, and phosphorylation in certain tumor cells have influenced the development of radiolabeled choline derivatives as diagnostic probes for imaging cell membrane proliferation. We, therefore, aimed to develop a choline-based moiety for imaging choline kinase-overexpressed tumors by single-photon emission tomography (SPECT). A novel choline-based diagnostic probe was synthesized and evaluated preclinically in various ChT- and ChK-overexpressed tumor models for SPECT imaging applications. Methods: The synthesis of diethylenetriaminepentaacetic acid-bis-choline ethylamine [DTPA-bis(ChoEA)] featured the conjugation of dimethylaminoethanol to a bifunctional chelator DTPA anhydride. [99mTc]Tc-DTPA-bis(ChoEA) was prepared, and its in vivo characteristics were evaluated in BALB/c mice and tumor-xenografted PC3, A549, and HCT116 athymic mouse models. The in vitro parameters, including cell binding and cytotoxicity, were assessed in PC3, A549, and HCT116 cell lines. To evaluate the specificity of the radioprobe, competitive binding studies were performed. Small-animal SPECT/CT diagnostic imaging was performed for in vivo evaluation. The mouse biodistribution data was further investigated to estimate the radiation dose in humans. Results: In silico studies suggested high binding with enhanced specificity. A standard radiolabeling procedure using stannous chloride as a reducing agent showed a labeling yield of 99.5 ± 0.5%. The in silico studies suggested high binding with enhanced specificity. [99mTc]Tc-DTPA-bis(ChoEA) showed high in vitro stability and specificity. The pharmacokinetic studies of [99mTc]Tc-DTPA-bis(ChoEA) in mice showed an increased tumor-to-background ratio after few minutes of intravenous administration. The first-in-human trial was also conducted. The effective dose was estimated to be 0.00467 mSv/MBq (4.67 mSv/GBq), resulting in a radiation dose of up to 1.73 mSv for the 370 MBq injection of [99mTc]Tc-DTPA-bis(ChoEA). Conclusions: The synthesized radioprobe [99mTc]Tc-DTPA-bis(ChoEA) accumulates specifically in choline kinase-overexpressed tumors with a high signal-to-noise ratio. The preclinical and first-in-man data suggested that [99mTc]Tc-DTPA-bis(ChoEA) could potentially be used as a diagnostic SPECT tracer in the monitoring and staging of cancer.