Background: Human epidermal growth factor receptor 2 (HER2) overexpression is related to many tumor treatments. RC48-ADC, a novel humanized anti-HER2 antibody conjugated with monomethyl auristatin E, has shown a promising efficacy in patients with HER2-positive locally advanced or metastatic urothelial carcinoma (mUC). The characteristic expression and scoring systems of HER2 have existed in breast cancer, gastric cancer for many years, but not in UC. We aimed to explore the expression pattern of HER2 in UC and develop a validated HER2 scoring system. Methods: A total of 137 patients and 43 patients in two studies of cohort 1 and cohort 2 were enrolled, respectively. The patients of the cohort 2 were enrolled in the open-label, multicenter, phase II study of RC48-ADC. Formalin-fixed paraffin-embedded urothelial cancer samples were tested for HER2 status using the fluorescence in situ hybridization (FISH) PathVysion HER2 DNA probe kit (PathVysion, Abbott Molecular, USA). Immunohistochemistry (IHC) was performed using the Ventana Benchmark XT (Ventana Medical Systems, USA). The 2018 ASCO/CAP HER2 scoring system of breast cancer was adopted and modified to score HER2 expression level in UC. Results: The expression rate of HER2 (IHC 2+/3+) was 24.1% (33/137). In HER2 IHC status 3+ or 2+ patients, the HER2 amplified rate was 31% (13/42). The objective response rates in RC48-ADC treatment patients with IHC 3+, IHC 2+ and FISH +, IHC 2+ and FISH - were 58.8%, 66.7% and 40%, respectively. Heterogeneity of HER2 protein expression was 55.5% (15/27) and the objective response rate had no significant difference between patients with tumor heterogeneity and homogeneity. Conclusions: The modified HER2 testing scoring system could be applied to UC to determine which patient might benefit from anti-HER2-ADC treatment. There was a trend towards a better benefit for patients with HER2 amplification and the tumor heterogeneity did not influence the drug efficacy.