A novel peak-differentiation-imitating assisted SERS (PDI-SERS) method is proposed for the first time to differentiate estrone, 17β-estradiol, and estriol utilizing plasmonic MIL-101(Fe) nanoparticles. Our PDI-SERS strategy enables the discrimination of subtle differences in the SERS spectra of phenolic estrogens, even in the same chemical environment, offering an important paradigm for molecular differentiation and accurate quantification in complex matrices.