The main purpose of this investigation was to detect the variation among sequential tissue culture stages of sugarcane varity GT54- C9. In order to achieve such a purpose, peroxidase isozymes and total protein patterns were studied and used as a tool to detect these differences during the different stages of tissue culture. The obtained results indicated that, significant differences among different tissue culture stages for sugarcane varity GT54- c9 were observed and detected as a result of presence or absence of some bands among the different tissue culture stages or the presence the same bands as a homo or heterozygous band then turned to be in another form. Based on these observations, different bands were observed for peroxidase isozymes patterns among the different stages of tissue culture technique. also, different protein patterns for sequential stages of tissue culture technique which applied on sugarcane varity gt54- c9 were confirmed. 90, 73, 65, 20, 19.7, 19.5, 18.8,11 and 8 kda could be considered a differential protein bands among the different stages of tissue culture to MS basal medium, while the addition of high concentration of sucrose (80 g ∕ L) found to be ineffective factor for root regeneration and growth. Micropropagation system was performed by exposure of axillaries and terminal buds to solid MS medium which contained 0.1 IAA and 1.0 BA. Then, clusters were cut into small number of plantlets in MS liquid media without any hormone. This method was found to be more effective than using hormonal medium for micropropagation.