Pre-clinical diagnosis of many diseases required quantitative detection of Human Serum Albumin (HSA). Herein a high-selective HSA sensor RhHSA was picked through a two-round selectivity evolution from the typical "Effector-π-Trigger" style. RhHSA suggested advantages including high selective (∼6 fold for HSA:BSA = 1:10), sensitive (LOD ∼ 5 nM, over 700-fold enhancement), steady (over 24 h) and wide linear range (0–0.5 mg/mL, applicative for conventional HSA measurement). The detecting system was free from media polarity or viscosity. HSA destruction, site competition and molecular docking provided reliable evidence for the fact that RhHSA could be embedded into both ibuprofen and phenylbutazone sites of HSA. These hints also supported the discrimination of HSA from BSA. Stepwisely fluid replacement in living cells and measuring in urine system both inferred the potential of RhHSA in biological applications. Display omitted •Selectivity evolution has been conducted for the first time in seeking HSA sensors.•RhHSA showed fine properties, especially the top class discrimination ability of HSA from BSA.•Pioneering stepwisely fluid replacement in living cells to evaluate the depletion of different albumin.•Detection mechanism hinted by HSA destruction, site competition and molecular docking.