Heavy single-chain antibodies (VHH or nanobodies) are popular in the medical and analytical felds due to its small size, high solubility, stability, and other advantageous features. However, the usage of VHHs is limited by the low yield of its production and purifcation. In order to determine the optimal purifcation strategy for VHH to improve the yield, a method to monitor purifcation at the intermediate steps is needed. In this study, a simple, sensitive, low-cost sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantitate VHHs throughout the purifcation steps. Under optimized conditions, the assay has a sensitivity of 0.149 OD·mL/ng and a limit of detection (LOD) of 0.029 ng/mL. The average recoveries of the assay against the spiked samples were 101.9–106.0% and 100.7–108.0%. The method was applied to a variety of real samples for the detection of diferent VHHs in bacterial cell media. High amount of VHHs (up to 41.3 mg/mL), which are comparable to the average yield of VHH in standard production protocols, were detected in the media. This study raises attention to the problem of protein losses in cell culture supernatants and provides a method for the continuous detection of the protein abundance to optimize the expression and purifcation protocols especially for nanobodies.