The molecular formation mechanism of 2-furfurylthiol in the glucose-cysteine reaction is not reported. Knowledge of the molecular interaction of glucose and ribose on the generation of 2-furfurylthiol is still unclear. The carbon module labeling (CAMOLA) technical approach was applied to elucidate the formation mechanism of 2-furfurylthiol in the glucose-cysteine reaction. The effect of ribose on the glucose-cysteine reaction was also evaluated. The results showed that 2-furfural and 2-furanmethanol were important intermediates for the formation of 2-furfurylthiol. Irrespective of the heating time, 2-furfurylthiol was mainly generated from an intact C5 glucose skeleton (88–89%), whereas the recombination of glucose fragments had minimal contribution. 2-Furfural could be generated from the Maillard reaction between glucose and cysteine or glucose alone, which further formed 2-furanmethanol. Immediately, 2-furfurylthiol could arise from the reaction of 2-furanmethanol and H2S from cysteine. Moreover, the reaction of glucose, ribose, and cysteine could generate 2-furfural, 2-furanmethanol, and 2-furfurylthiol by an addition effect confirmed by the model reaction and food system.