This work describes the design of optical aptamer-based porous silicon (PSi) biosensors for the direct capture of Lactobacillus acidophilus. Aptamers are oligonucleotides (single-stranded DNA or RNA) that can bind their targets with high affinity and specificity, making them excellent recognition elements for biosensing applications. Herein, aptamer Hemag1P, which specifically targets the important probiotic L. acidophilus, was utilized for direct bacteria capture onto oxidized PSi Fabry–Pérot thin films. Monitoring changes in the reflectivity spectrum (using reflective interferometric Fourier transform spectroscopy) allows for bacteria detection in a label-free, simple and rapid manner. The performance of the biosensor was optimized by tuning the PSi nanostructure, its optical properties, as well as the immobilization density of the aptamer. We demonstrate the high selectivity and specificity of this simple “direct-capture” biosensing scheme and show its ability to distinguish between live and dead bacteria. The resulting biosensor presents a robust and rapid method for the specific detection of live L. acidophilus at concentrations relevant for probiotic products and as low as 10⁶ cells per mL. Rapid monitoring of probiotic bacteria is crucial for quality, purity and safety control as the use of probiotics in functional foods and pharmaceuticals is becoming increasingly popular.