► A new SPR platform ‘Phytochip’ was developed and established. ► It provides an improved detection assay to distinguish virus-infected plants from non-infected plants. ► It has a short detection time and can be used in a high throughput system to analyze efficiently many samples. ► Phytochip is suitable for practical application in plant breeding and virus control. The surface plasmon resonance (SPR) based ‘Phytochip’ was developed to distinguish virus-infected plants from non-infected plants. The system detects DNA–RNA hybridization to show the presence of phytopathogenic viruses such as the RNA virus barley stripe mosaic virus (BSMV) in wheat leaves. To achieve this BSMV and wheat specific oligonucleotides, and a negative control yeast oligonucleotide, were immobilized on a SPR gold surface chip. After optimization of the hybridization parameters with purified wheat samples, wheat infected with BSMV resulted in detectable signals with both the BSMV and the wheat probes. In contrast, a hybridization reaction was not be detected with the negative probe. The method is fast and sensitive with a detection time of 3000s (50min), a detection limit of 14.7pgμl−1 BSMV RNA and a measuring range of 14.7–84pgμl−1 BSMV RNA (1.323–7.56ng BSMV RNA per 90μl sample). These characteristics, combined with the high throughput design, make it suitable for application in plant breeding and virus control.