Cell adhesion plays an important role in cell morphology, motility, and differentiation, and cancer metastasis. The rigidity of the substrates is one of the factors that can affect cell behaviors. How the rigidity of substrates influence the initial attaching strength of cell is not well investigated yet. Cell probe attaching cell on the AFM cantilever tip is a new approach to study cell mechanics. The purpose of this study is to investigate the relation between the rigidity of substrate and cell detaching force by cell probe. Fibroblast cell was used to quantify cell detaching force. All the surfaces of substrates were coated with type I collagen. The contact times were set at 30, 60, 90, and 300 seconds with contact compression at 500 pN. The substrates used were 1037 Pa, and 10930 Pa poly acrylamide (PAA) and glass. Under the same contact time, the detaching force rose gradually with increasing substrate rigidity. They were 482.11±194.34, 1820.11±949.29, and 3373.45±1867.02 pN respectively for 1037 Pa, and 10930 Pa PAA and glass for 300 seconds. Our results showed cell adhesion could be influenced by substrate rigidity. This cell probe technique can be further used for parameter studies on cell substrate interaction in the future.