Thiols play a crucial role in various biological processes, and the discrimination of thiols in biofluids is a significant but difficult issue. Herein, a facile label-free non-invasive fluorescent sensor array has been presented based on PDA/PEI_n–Cu²⁺ in three different pH buffer solutions for pattern discrimination of thiols and chiral recognition of cysteine (Cys) enantiomers in biofluids toward health monitoring. The proposed sensor array was fabricated based on the fact that Cu²⁺ has a strong affinity toward thiols, which prevents Cu²⁺ from binding PDA/PEI_n, and the fluorescence properties of PDA/PEI_n were recovered to a certain degree. Different thiols exhibited different affinities toward Cu²⁺, generating distinct fluorescence response patterns. These response patterns are characteristic for each thiol and can be discriminated by principal component analysis (PCA). In this work, three types of PDA/PEI₄₈–Cu²⁺ sensors (PDA/PEI₄₈–Cu₄²⁺, PDA/PEI₄₈–Cu_4.5²⁺ and PDA/PEI₄₈–Cu₅²⁺) were prepared by using acetate buffer with different pHs (at 4, 4.5, and 5) to form our proposed sensor array, which could realize the pattern discrimination of 8 thiols. Moreover, we successfully realized the sensitivity and selectivity assays to these thiols. Furthermore, the proposed sensor array could discriminate mixtures of thiols as well as the chiral recognition of mixtures of Cys enantiomers, promising its potential practical usage. Significantly, the resultant practical application in real samples showed that it could be a fascinating assay for the development of non-invasive diagnosis. This method promises the facile, sensitive and powerful discrimination of thiols in biofluids and would sprout more relevant strategies toward a broad range of applications.