Although carbon dots (CDs) as fluorescent sensors have been widely exploited, multi-component detection using CDs without tedious surface modification is always a challenging task. Here, two kinds of nitrogen-doped CDs (NCD-m and NCD-o) based on soluble starch (SS) as carbon source were prepared through one-pot hydrothermal process using m-phenylenediamine and o-phenylenediamine as nitrogenous dopant respectively. Through fluorescence “on-off” mechanism of CDs, NCD-m and NCD-o could be used as a fluorescence sensor for detection of Fe 3+ and Ag + with LOD of 0.25 and 0.5 μM, respectively. Additionally, NCD-m could be used for indirect detection of ascorbic acid (AA) with LOD of 5 μM. Moreover, fluorescence intensity of NCD-m also exhibited the sensitivity to pH change from 2 to 13. More importantly, Both NCD-m and NCD-o had potential application for analysis of complicated real samples such as tap water, Vitamin C tablets and orange juice. Ultimately, the small size of NCD-m could contribute to reinforcing intracellular endocytosis, which allowed them to be used for bacteria imaging.