The tenderness of the fresh tea leaves can affect the quality of tea products. It is important to develop a mechanized, accurate way to evaluate the quality of fresh leaves that avoids the uncertainty of a subjective evaluation. Herein, an in-situ, ultra-rapid Raman microscopy strategy to quantify carotenoids in tea leaves was established. The Raman microscopy of carotenoids distribution in leaves from new branches of 22 representative tea varieties showed that the average carotenoid signals increased from a low level in the bud to a high level in the fourth leaf, which represent different developmental stages. The concentration of carotenoids in the bud to fourth leaf, which were from 69.1 ng mg−1 to 199.5 ng mg−1, respectively. These results demonstrate that Raman imaging can serve as an in-situ, non-destructive and ultra-rapid technology for determining the tenderness of fresh tea leaves and be used in quality control for tea processing.