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Background: Sweet sorghum (Sorghum bicolor L. Moench) has the potential as an alternative of bioethanol feedstock without impacting food security. The crop can adapt widely to diverse environments and altitudes and be able to produce on marginal land. The ratoon crop of sweet sorghum also followed the same trend to the main plant. According to Tsuchihashi and Goto (2008), sorghum plants can be harvested two to three times, including main and ratoon crops, so that it can be as raw materials of bioethanol in a sustainability. The sugar content of sweet sorghum stems ranges from 14 % to 23 % (Almodares and Hadi, 2009) and in the second harvest was reduced 20 % ( Mingli, et.al., 2012). Further, to increase the yield, application of biofertilizers in adequate quantities is required. Four sweet sorghum varieties were evaluated in a main and ratoon cropping system 800 m asl (above sea level) during April 2016- January 2017. Aims: The study was conducted to compare the sugar content, juice production, and total biomass between primary and ratoon crops some variety of sweet sorghum in highlands. Materials and Methods: This study had been performed in highlands location was Trosono Village in Parang District of Magetan Regency, Indonesia, with an altitude of 800 m above sea level (asl). This experiment uses a factorial design with three replications. The size of the plot is 3.5 m × 1.5 m and planting distance between rows and within a row are 70 cm and 20 cm respectively. Five plants in every treatment were randomly chosen to be measured. The factors have represented the dosage of biofertilizer are 5 g ∙ plant –1, 10 g ∙ plant –1, 15 g ∙ plant–1, and varieties of sweet sorghum are Suri 3, Kawali, Super 2, and Suri 4. The biofertilizers contain Trichoderma sp, Pseudomonas fluorescence, Bacillus subtilis, and indigenous mycorrhizae. Analysis of variance was undertaken using SPSS version 25, and treatment average separation was conducted using Duncan-s Multiple Range Test implemented in SPSS. Results: The result obtained from Duncan-s Multiple Range Test showed that the sugar content of sweet sorghum all varieties was no significant difference among the means in ratoon crops. However, the sugar content showed an increase compared to main crops. Meanwhile, the volume of juice extraction has decreased in ratoon crops. Figure 1 and 2 showed that the Suri-4 variety increases sugar content and also reduce juice production the lowest. Conclusion: Varieties Suri-4 used as feedstock for bioethanol better than other variety since has decreasing of the sugar content lowest in ratoon crops. References 1. Almodares, A. and M.R. Hadi. 2009. ‘Production of bioethanol from sweet sorghum-: A review. African J. Agri. 4(9), pp. 772-780. 2. Mingli W., Zhanguo X., Brandon T., Glenn F., David P., Zhenbang C., Jerry D., Jianming Y., Yen-Con H., Gary A. P. 2012. ‘Evaluation of Sweet Sorghum as a Feedstock by Multiple Harve