A process for isolation and purification of TNK-tPA comprising steps of: (i) subjecting cell free harvest obtained from CHO cell culture to affinity chromatography to capture TNK-tPA and obtaining an eluate containing partially purified TNK-tPA; wherein the affinity chromatography comprises Blue Sepharose 6 FF as a stationary phase and a mobile phase, wherein the mobile phase is a mixture of sodium phosphate buffer, sodium chloride and urea; (ii) subjecting the eluate of step (i) to affinity chromatography for additional purification of TNK-tPA and obtain an eluate containing primarily TNK-tPA; wherein the affinity chromatography comprises Lysine Hyper D as a stationary phase and a mobile phase, wherein the mobile phase is a mixture of sodium acetate buffer, urea and epsilon aminocarproic acid (EACA); (iii) viral inactivation of eluate of step (ii) to obtain the viral inactivated sample; wherein viral inactivation is conducted by low pH and chemical inactivation, wherein chemical inactivation is done by treating the sample with sodium caprylate in the presence of urea; (iv) subjecting the viral inactivated sample of step (iii) to a further affinity chromatography-III for additional purification to obtain an eluate containing primarily highly purified TNK-tPA; wherein the affinity chromatography comprises is Ceramic Hydroxy Apatite as a stationary phase and a mobile phase, wherein the mobile phase is a mixture of phosphate buffer, 2-(N-Morpholino) ethanesulfonic acid hydrate (MES- Hydrate), sodium chloride and urea; (v) subjecting the eluate of step (iv) to cation exchange chromatography to obtain an eluate containing highly purified preparation of TNK-tPA; wherein cation exchange chromatography comprises Fractogel SO 3 as a stationary phase and a mobile phase, wherein the mobile phase is a mixture of L-Arginine, O-phosphoric acid and polysorbate-20; (vi) subjecting the eluate of step (v) to virus reduction filtration for removal of virus present; wherein virus filtration is carried out by Polyethersulphone nanofilter having a size in a range from 15-20 nm; (vii) concentrating the sample of step (vi) to obtain TNK- tPA by tangential flow filtration; wherein the yield of the process is more than 60 % and purity of TNK-tPA obtained is more than 95% as measured by Size exclusion chromatography. (i) subjecting cell free harvest obtained from CHO cell culture to affinity chromatography to capture TNK-tPA and obtaining an eluate containing partially purified TNK-tPA; wherein the affinity chromatography comprises Blue Sepharose 6 FF as a stationary phase and a mobile phase, wherein the mobile phase is a mixture of sodium phosphate buffer, sodium chloride and urea; (ii) subjecting the eluate of step (i) to affinity chromatography for additional purification of TNK-tPA and obtain an eluate containing primarily TNK-tPA; wherein the affinity chromatography comprises Lysine Hyper D as a stationary phase and a mobile phase, wherein the mobile phase is a mixture of sodium acetate buffer, urea and epsilon aminocarproic acid (EACA); (iii) viral inactivation of eluate of step (ii) to obtain the viral inactivated sample; wherein viral inactivation is conducted by low pH and chemical inactivation, wherein chemical inactivation is done by treating the sample with sodium caprylate in the presence of urea; (iv) subjecting the viral inactivated sample of step (iii) to a further affinity chromatography-III for additional purification to obtain an eluate containing primarily highly purified TNK-tPA; wherein the affinity chromatography comprises is Ceramic Hydroxy Apatite as a stationary phase and a mobile phase, wherein the mobile phase is a mixture of phosphate buffer, 2-(N-Morpholino) ethanesulfonic acid hydrate (MES- Hydrate), sodium chloride and urea; (v) subjecting the eluate of step (iv) to cation exchange chromatography to obtain an eluate containing highly purified preparation of TNK-tPA; wherein cation exchange chromatography comprises Fractogel SO 3 as a stationary phase and a mobile phase, wherein the mobile phase is a mixture of L-Arginine, O-phosphoric acid and polysorbate-20; (vi) subjecting the eluate of step (v) to virus reduction filtration for removal of virus present; wherein virus filtration is carried out by Polyethersulphone nanofilter having a size in a range from 15-20 nm; (vii) concentrating the sample of step (vi) to obtain TNK- tPA by tangential flow filtration; wherein the yield of the process is more than 60 % and purity of TNK-tPA obtained is more than 95% as measured by Size exclusion chromatography.