Carbon dots are admirable fluorescent nanomaterials due to their low cost, high photostability, excellent biocompatibility, and environmental friendliness. Most conventional carbon dot fabrication approaches produce single-colored fluorescent material in the preparation process; different methods are therefore required to synthesize distinct carbon dots for specific optical applications. In this study, carbon dots carrying different emission colors are prepared through a one-step refluxing process. The emission of these materials can be well-tuned by sodium hydroxide content in the precursor solution. The carbon dots produced are used as sensing probes based on the spectrofluorometric inner filter effect for target molecule detection. Three sensing categories that combine carbon dots and inner filter effect are demonstrated, including direct, metal nanoparticle-assisted, and enzymatic reaction-supported detection. Caffeine, melamine, and fenitrothion are selected as targets to demonstrate the strategies, respectively. These multifunctional carbon dot-based sensors achieve comparable sensitivity toward analytes with a much more convenient preparation route.