A selective, rapid, small sample load (2–4 μL), and sensitive quantification method for the hydrophobic cellular biomolecules of pathogenic bacteria and their biosensing application were reported. The present approach is based on using polythiophene polymer dots (2.5 nm), which were prepared via the oxidation/polymerization reactions and then were characterized using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR), ultraviolet spectroscopy (UV), and matrix (surface) assisted laser desorption/ionization mass spectrometry (M(S)ALDI-MS). The present method requires only gentle agitation for a single drop of aqueous bacteria suspension (10 μL, Pseudomonas aeruginosa (1 × 104 cfu/mL) and Staphylococcus aureus (1 × 105 cfu/mL)) with 1 mL of polythiophene (0.5 mg/mL) in chloroform, and the time required for quantifying the total hydrophobic was significantly reduced to less than 3 min. The polythiophene polymer dots is also a quantitative assessment of bacteria for aqueous and blood samples if exposed to more than 4–5 μL of pathogenic bacteria and thus, it is a new biosensor for quantitative hydrophobic portions. The fluorescence intensity of polythiophene was enhanced after adding different volumes of pathogenic bacteria with low colony units. The standard bacteria suspensions of P. aeruginosa and S. aureus have low LOD (limits of detection) for 2 μL (1 × 104 cfu/mL) and 4 μL (1 × 105 cfu/mL), respectively. Further, the pathogenic bacteria were spiked into mouse blood and the total hydrophobic biomolecules were quantified. This method is extremely rapid as it does not require any culture steps prior to analysis and also no need for any separation or post sample treatments.