The proposed research specifically addresses the most prevalent health problem in dairy cows, the bovine mastitis (BM), by developing a generic biosensing platform for on-site monitoring applications. Traditional diagnostic methods check the quality of milk through the detection of mammary gland inflammation, infection diagnosis and its causative pathogens. However, all those techniques lack the ability to detect analyses in real-time or outside the laboratory boundaries. Therefore, there is an urgent need for a rapid, accurate, cost effective, simple and portable method to evaluate in real-time new cases of BM, followed by corrective and preventive actions, leading to a positive effect on animal health and overall economy of the dairy farms. Herein, we have designed and fabricated an optical biosensing platform, based on porous silicon nanostructures, Fabry-Pérot film, for the detection of BM-predicting biomarkers, haptoglobin (Hp) and N-acetyl-β-d-glucosaminidase, which are monitored in real-time by reflective interferometric Fourier transform spectroscopy. Indeed, increased Hp levels cause a profound increase in the relative optical signal as specific hemoglobin-Hp complexes are formed. Thus, for clinical and subclinical BM values a red shift of 0.36 ± 0.04% and 0.24 ± 0.006% in the effective optical thickness are obtained. These results correlate to Hp concentrations obtained by ELISA test in which values of 9.6 ± 0.3 and 2.3 ± 0.1µg/mL are received for clinical and sub-clinical milk samples (both positive to S. dysgalactiae pathogen), respectively. Control experiment of healthy milk (SCC < 100,000, with negative microbial contamination) presented a minor increase of 0.06% in optical output as insignificant values of Hp are obtained (1.1 ± 0.1 µg/mL). Similar differentiation is shown for the latter biomarker. Our studies reveal that the optical nanostructure capable of multitasking: immobilization, size-exclusion, rapid and sensitive detection of specific biological targets in real milk samples, offering simultaneous real-time detection based on physical characteristics. Thus, the main advantage of the presented biosensing concept is the ability to detect BM predicting biomarkers in milk, using a simple and portable experimental setup.