Microbial fuel cell (MFC) technology can be potentially developed as an alternative energy source since it can convert various substrates from renewable sources into electricity using bacteria as biocatalyst. Tempe wastewater as MFC substrate gives advantages in tempe wastewater treatment and reducing the purchasing cost of bacteria. Currently, the applications of MFCs are still limited due to the relatively low electricity production, so many studies have been conducted to improve the electricity production by MFC. This study focused on investigating the influence of biofilm formation time and the use of macromolecule as additional substrate towards electricity production from MFC system with tubular membranless reactor and tempe wastewater as substrate. This study suggested that biofilm formation on anode could improve the electricity production up to 10-folds while the use of glucose as substrate addition reduce the electricity production up to 60%. The biggest electricity output was obtained from the experiment of biofilm formation for 14 days with EPS content in biofilm 0,13 mg/cm2 where the maximum voltage and power density produced was respectively 34,81 mV and 0,26 mW/m2.