Although a few of fluorescent probes based on carbon dots (CDs) for vitamin B (VB) determination have been emerged, none of them can realize the detection of different kinds of VB. In this paper, nitrogen, chlorine co-doped dual-emission CDs (N, Cl-CDs) with emissions at 404 nm and 595 nm have been easily synthesized. VB2, VB9 and VB12 can all induce obvious fluorescence turn-off response toward the N, Cl-CDs. Based on that, three types of VBs are quantitatively and sensitively evaluated in aqueous solution with wide concentration ranges of 14.9–135.0 μM, 34.7–89.8 μM and 29.8–79.8 μM, respectively. Importantly, visual semiquantitative detection of VBs on a test strip are also proposed. Moreover, the current N, Cl-CDs have been successfully applied to the detection of VBs in real samples. The N, Cl-CDs are sensitively multifunctional sensors for three kinds of VBs in aqueous solution and the visual semiquantitative detection by test paper assay is simple, portable and inexpensive.