The fermentative study on optimization of lactic acid production from cane sugar molasses by isolated Lactobacillus bacteria have been carried out to assess the most suitable condition for its production and to obtain high yield. The study on the optimization effect of different process parameters such as pH of the medium, temperature, inoculums size, incubation time and agitation rate were done in order to enhance the conversion of cane sugar into lactic acid. Optimization was done for Initial pH, temperature, inoculum size, incubation time and agitation rate. The optimum condition was found for fermentation with the process conditions of pH (8.0) temperature (40 °C) and inoculum size (7% (v/v)) with an incubation time of 144 h and effective agitation speed of 175. The above mentioned optimized process parameters can be used in large scale production of lactic acid fermentation in further investigations by using cane sugar as a substrate .