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During the past decade, increasing attention has been paid to fluorescent carbon dots (CDs) due to their unique photoluminescence (PL) properties. As synthetic methods gradually develop, many post‐functionalization strategies have been developed to enhance the PL of CDs. However, in most cases, the PL enhancement was less than 10‐fold with multistep modification processes. In this work, a facile and efficient post‐functionalization strategy was successfully applied to enhance the PL intensity of CDs dramatically up to 69 times by surfactants at room temperature for the first time. Furthermore, the mechanism of surfactant‐induced PL enhancement of CDs was investigated and in vivo bioimaging was performed. The results demonstrated that electrostatic/non‐electrostatic interactions between CDs and surfactants could effectively lower the vibration and rotation of CDs, increase radiative decay processes and, thus, enhance the PL of CDs. This finding may provide new insights into the strategies for enhancing the PL of CDs, further broadening their potential applications. Brighter Carbon Dots: The effect of different types of surfactants on the photoluminescence of carbon dots (CDs) was investigated. The results show that interactions between carbon dots and surfactants enhance the photoluminescence of CDs. [ABSTRACT FROM AUTHOR]