<P><B>Abstract</B></P> <P>The effect of glycerol waste (GW) concentration was studied in anaerobic co-digestion with canned sardine wastewater (CSW), has been examined using the 1-stage mesophilic (P1), 1-stage thermophilic (P2) and 2-stage mesophilic (P3) process. P3 process was the best process, could improve the biogas production (bio-hythane) in the case of canned sardine industry. Anaerobic batch co-digestion between 4%GW (v/v) and CSW in P3 had the maximum yield of hydrogen and methane. The hydrogen and methane yield were 43.00 ml H<SUB>2</SUB>/g COD<SUB>r</SUB> and 303.69 ml CH<SUB>4</SUB>/g COD<SUB>r</SUB>, respectively. Total hydrogen and methane production increased 648.57 and 7.75 fold compared with the single-digestion of CSW at 5 and 45 days of fermentation. The total energy yield in bio-hythane formation was 8.07 MJ/kg COD with consisting 43.11% CH<SUB>4</SUB>, 21.45%H<SUB>2</SUB> and 35.43% CO<SUB>2</SUB>. Using GW as co-substrate was the synergism for increasing the potential to produce hydrogen and methane (bio-hythane) in CSW.</P>