Restriction Endonucleases play a major role in recombinant DNA technology.Various protocols have been developed for the purification,involving different chromatographic steps.We have exploited the applicability of one step chromatography in the purification of EcoR Ⅰ.the method is effective and economical.In this method E.coli RY13,was grown at 37℃ in 2 liters LB media at 360 rpm agitation and l vvm aeration to late log phase.The cell harvested by using centrifuge with 3000 rpm during 20 min.the pellet suspended in lyses buffer,sonicated and centrifuged at 100000g for Ⅰ h.the supematant was applied on p11 column and eluted with linear gradient of 0.2M to 0.8 M NaCL in buffer.The enzyme activity was eluted between 0.7 mol/L to 0.75 mol/L in NaCl.The active fractions were pooled and dialyzed against storage buffer and keep at-20 ℃.The enzyme was assayed by using standard protocol.