The invention relates to a preparation method of a fast-respondability Hg2+developing fluorescence probe based on a rhodamine B-thiophene derivative. The preparation method comprises the steps of performing a condensation acylation reaction by using rhodamine B and DETA (diethylenetriamine), so as to obtain the rhodamine B-DETA derivative, then reacting the rhodamine B-DETA derivative with 2-thiophenecarboxaldehyde, so as to obtain the rhodamine B-thiophene derivative, and a water-ethyl alcohol mixing solution based on the rhodamine B-thiophene derivative can be used for the fast-respondability Hg2+developing fluorescence probe. The preparation method provided by the invention can be used for the fast-respondability developing fluorescence probe for rapidly analyzing the content of Hg2+ in a water sample, and the fast-respondability developing fluorescence probe has strong anti-interference to other metal ions (Na+, K+, Mg2+, Fe3+, Cu2+, Cr3+), the applicable pH value range is from acid to neutral, and the fast-respondability developing fluorescence probe is an ideal Hg2+developing fluorescence probe; the respond time of an Hg2+ color indicator to the Hg2+ is less than 2 minutes, the lowest detectable limit is 2.0 times 10<-7> mol/L; the preparation method provided by the invention has the advantages of being simple in synthetic process, low in cost, easy and simple to operate, high in sensitivity, quick in respond speed, low in detectable limit, wide in application prospect and the like.