The present invention relates to a biochip for the detection of phosphorylation and a detection method of phosphorylation using the same, more precisely a biochip prepared by integrating a recombinant fusion protein produced from the reaction of a kinase matrix selected from the group consisting of PKC (Protein Kinase C), cc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase) and the elevated protein Selenomonas ruminantium membrane protein on a substrate coated with an active group, a detection kit of phosphorylation composed of the said biochip and a cofactor labeled with a radio-isotope and a detection method of phosphorylation using the same. The biochip for the detection of phosphorylation of the present invention using a radio-isotope facilitates the detection of phosphorylation with a minimum amount of a sample by simple processes, compared with the conventional method using an antibody. Since this method can analyze a large amount of samples in a shorter period of time, it can be effectively used for the analysis of kinase activity.