Daratumumab (DARA) is an anti-CD38 monoclonal antibody for the treatment of multiple myeloma (MM). The tumor CD38 expression level is one of the important factors in determining the efficacy of DARA treatment. Therefore, there is an urgent clinical need for a noninvasive tool to evaluate the CD38 levels in cancer patients before, during, and after DARA treatment. In this study, we prepared a new molecular imaging probe 99m Tc-CD3813, the 99m Tc-labeled nanobody CD3813, for noninvasive imaging of CD38 expression by single photon emission computed tomography (SPECT). We evaluated 99m Tc-CD3813 for its CD38 affinity and specificity and its capacity to image the CD38 expression in the MM and lymphoma xenografts models. 99m Tc-CD3813 SPECT/CT is able to visualize subcutaneous/orthotopic myeloma lesions in animal models and has advantages over 18 F-fluorodeoxyglucose ( 18 F-FDG) positron emission tomography. Excess DARA has less impact on its tumor uptake (3.14 ± 0.83 vs 2.29 ± 0.91 %ID/g, n.s.), strongly suggesting that there is no competition between 99m Tc-CD3813 and DARA in binding to CD38. 99m Tc-CD3813 SPECT/CT revealed significant reduction in CD38 expression in the Ramos-bearing mice under DARA treatment, as evidenced by their reduced tumor uptake (3.04 ± 0.70 vs 1.07 ± 0.28 %ID/cc, P < 0.001). 99m Tc-CD3813 SPECT/CT was also able to detect the increased tumor uptake (0.79 ± 0.29 vs 2.12 ± 0.12 %ID/cc, P < 0.001) due to the upregulation of CD38 levels caused by all-trans retinoic acid infection. 99m Tc-CD3813 is a promising SPECT radiotracer for imaging the CD38-positive tumors and has clinical potential as a molecular imaging tool for evaluation of the CD38 expression level in patients before, during, and after DARA treatment.