Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI) is rapidly maturing as an innovative technique for spatial molecule ( m/ z > 1000 Da) profiling. However, direct identification of low-molecular-weight compounds ( m/ z < 600 Da) by MALDI-TOF MSI using conventional organic matrixes remains a challenge because of ionization suppression and serious matrix-related background interference. Furthermore, the heterogeneous cocrystallization that is inherent to organic matrixes can degrade spatial resolution in MSI. Herein, we developed a negative ion surface-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (SALDI-TOF MSI) protocol to detect bisphenol S (BPS) and map its spatial distribution in mouse tissues by applying nitrogen- and sulfur-co-doped carbon dots (N,S-co-doped CDs) as a new matrix through spraying. The SALDI-TOF MS and imaging parameters, such as matrix concentration, ionization mode, and matrix deposition, were optimized to improve imaging performance. In comparison to organic matrixes, the use of N,S-co-doped CDs in negative ion mode exhibited free matrix background interference, enhanced MS signal intensity, and provided high spatial resolution (acquired at ∼50 μm) in the analysis of BPS, which allowed sensitive detection of the target compound on the surfaces of tissue sections. Quantitative assessment was also made by spotting BPS standards directly onto the tissue surface, and a good correlation between the color change and BPS concentrations was found. The corresponding detection limit as low as the ∼pmol level for BPS was observed with the direct visualization from MS images. Furthermore, the feasibility of the proposed SALDI-TOF MSI method was extended for in situ identification of exogenous BPS in the different tissues of mouse involving liver, kidney, spleen, and heart for exposure and profiling its spatial localization at different administration times. In addition, the general applicability of the proposed method was also evaluated by SALDI-TOF MSI analysis of BPAF in tissues. These successful applications of SALDI-TOF MSI not only demonstrated its promising potential as an alternative to MALDI-TOF MSI in profiling small molecules in tissue sections but also provided tremendous insight into the assessment of BPS exposure.