The fluorescence signal of Sybr Green can represent the amount of target DNA copy numbers in the samples, thereby determine a positive or negative diagnostic results after polymerase chain reaction (PCR). For precision quantification of the target gene in the sample, the fluorescent signal should be recorded in each cycle. Traditionally, highly sensitive devices such as silicon photomultipliers and high-power light source such as laser diode were used to excite and detect the fluorescence from double strand DNA selective dyes. However, the costs of these components were too high to be widely applied for rapidly on-site screening of outbreak causing pathgens in local clinics and fields of tests. In this study, low-cost optical element such as LED and photodiode were used to implementan off-plane excitation method to enhance the fluorescence signal. Several parameters such as illumination angle between LED and photodiode in horizontal direction and tilted illumination in vertical direction were optimized in the experiment to ensure the performance. The results showed that the fluorescence intensity can be enhanced approximately 25 %. These results indicated that the developed illumination architecture had advantages of low background noise, high sensitivity and high fluorescence variation. This research has successfully demonstrated an off-plane excitation method for enhancing the resolution of qPCR system and applying in medical diagnostics, rapid screening and related applications.