Superoxide generated during the early imbibition is an excellent marker for evaluating seed vigor. In this paper, a new principle biosensor for non-invasive detection of seed vigor based on quantitative mea-surement of superoxide via selective probe 2-rnethyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2a] pyrazin-3-one (MCLA)-mediated chemiluminescence (CL) was developed. The biosensor, which used a compact single-photon counting module (SPCM) to collect the CL signal, could evaluate seed vigor in vivo. Benefiting from the high CL efficiency of MCLA reacting with superoxide and high sensitivity of the SPCM technique, the trace superoxide generated by dry seeds under storage state can be detected to achieve rapid and non-invasive determination of the seed vigor. In comparison with the traditional methods for fast measuring seed vigor based on measurement of physiological and biochemical properties. our pro-posed technique has significant advantages such as low cost, simplicity, convenient operation and short time consuming. To demonstrate the utility of the SyStem, it was applied to evaluate MCLA-mediated CL of three different plant species wheat (Ze Yu No.2), maize (Tai Cu No.1 and 2) and rice (Jing Dao No.21) seeds with different degrees of aging. The experimental results suggested that there was an excellent pos-itive correlation between the seed vigor assessment from quantitative TTC-test and the detection based on MCLA-mediated CL of superoxide measurement. The new principle of seed vigor measurement is a challenge and breakthrough to conventional method of seed vigor determination and may be a potential technique of the next generation seed vigor detection.