Due to the instrument designed by using a common surface plasmon resonance biosensor is extremely expensive, we established a portable and cost-effective surface plasmon resonance biosensing system. It is mainly composed of laser line generator, P-polarizer, customized prism, microfluidic cell, and line Charge Coupled Device (CCD) array. Microprocessor PIC24FJ128GA006 with embedded A/D converter, communication interface circuit and photoelectric signal amplifier circuit are used to obtain the weak signals from the biosensing system. Moreover, the line CCD module is checked and optimized on the number of pixels, pixels dimension, output amplifier and the timing diagram. The micro-flow cell is made of stainless steel with a high thermal conductivity, and the microprocessor based Proportional-Integral-Derivative (PID) temperature-controlled algorithm was designed to keep the constant temperature (25 °C) of the sample solutions. Correspondingly, the data algorithms designed especially to this biosensing system including amplitude-limiting filtering algorithm, data normalization and curve plotting were programmed efficiently. To validate the performance of the biosensor, ethanol solution samples at the concentrations of 5%, 7.5%, 10%, 12.5% and 15% in volumetric fractions were used, respectively. The fitting equation ΔRU=-752987.265+570237.348×RI with the R-Square of 0.97344 was established by delta response units (ΔRUs) to refractive indexes (RI). The maximum relative standard deviation (RSD) of 4.8% was obtained.