Evanescent wave cavity ring-down spectroscopy (EW-CRDS) is used to observe the adsorption isotherm for hemoglobin (Hb) from controlled urine samples to assess the potential for rapid diagnosis in hemoglobinuria. The absorbance of Hb at 425 nm is monitored using an alexandrite laser-pumped, room temperature, LiF:F +2** color-center pulsed laser. A minimum absorbance detection level of 2.57×10 -4 is achieved, corresponding to a minimum detectable concentration of Hb in urea of 5.8 nM. A multilayered Hb biofilm is formed, and a minimum of eight layers are required to model the adsorption isotherm, allowing for cooperative binding within the layers and extending 56 nm into the interface. A binding constant for Hb to silica 18.23±7.58×10 6 M is derived, and a binding constant for Hb to Hb in subsequent layers is determined to be 5.631 ±0.432 ×10 5 M. Stoichiometric binding coefficients of 1.530±0.981 for layer one and 1.792±0.162 for subsequent layers suggest that cooperative binding both to the silica surface and between the layers of the biofilm is important. © 2005 Society of Photo-Optical Instrumentation Engineers.