The interaction between two single-stranded DNA (ssDNA) molecules as pairing to a double-stranded DNA (dsDNA) molecule is studied by the reflectometric interference spectroscopy (RIFS) technology.A nano-porous anode alumina membrane coated an Au layer is employed as the sensor substrate.The results indicate that when there are mismatched nucleotide bases,the effective optical thicknesses (OTeff) have obvious difference,and the changes of OTeff are connected with the sensor layer thickness and the effective refractive index.It is also demonstrated that the RIFS technique can be used to precisely detect the ssDNA molecules with individual base mismatched as pairing to dsDNA.