The invention discloses a two-dimension nuclear magnetic resonance detection method used for water-soluble metabolites of in-vitro cells, which particularly includes the steps of: 1) washing culturedcells with a frozen phosphate buffer solution, performing centrifugation, and collecting cell deposits; 2) adding a phosphate buffer solution containing sodium silicate to the cell deposits to extractthe water-soluble metabolites of the cell line; and 3) performing the two-dimension NMR detection to the water-soluble metabolite extract in the step 2) to obtain relative contents of the water-soluble metabolites. In the method, steps of cell pretreatment, extraction of the water-soluble metabolites and NMR detection are reasonably optimized, so that extraction and detection efficiency of the water-soluble metabolites are increased. The method achieves accurate measurement of change on levels of the water-soluble metabolites of the in-vitro cells.