The use of exogenous fibrolytic enzymes (EFE) technology to improve the utilization of fibrous feeds is a novel approach for enhancing feed utilization ruminants performance. The effects of EFE xylanase on in vitro fermentation characteristics of maize stover (MS), oat straw (OS) and sugarcane bagasse (SCB) were examined using an in vitro gas production (GP) technique. Three doses of xylanase plus EFE (Dyadic PLUS, Dyadic international, Inc., Jupiter, FL) in liquid form were used at 0 (control), 60 (low), 120 (medium) and 240 (high) µg/g DM of substrate. Ruminal GP, CH₄ and CO₂ concentrations were recorded at 2, 4, 6, 8, 10, 12, 14, 24, and 48 h of incubation at 2 methods of application (direct or 72-h pre-incubation). Separate ANOVA was performed for each feedstuff (maize stover, oat straw and sugarcane). Each experiment was laid down in a 2 (mode of EZ application either by pre-treatment or directly) × 3 (rates of application of 60, 120, and 240 µg xylanase/g DM) factorial plus one control (no enzyme additive) completely random design, for a total of 7 experimental treatments. The analysis was performed using the PROC MIXED of SAS for factorial arrangements of treatments plus one control. Increased (P < 0.05) GP at different incubation times was observed with EFE doses addition for the incubated fibrous feeds versus control. The pre-incubation versus direct addition of EZ increased GP of the 3 feeds. However, the direct addition of EFE improved (P < 0.05) DM, neutral detergent fiber (NDF), and acid detergent fiber (ADF) degradabilities. Substrates incubated with higher doses of EFE increased (P < 0.05) GP, DM, NDF, and ADF degradabilities versus low and medium doses. Enzyme decreased (P < 0.05) rumen pH of MS and SCB compared with control. Ammonia-N and total volatile fatty acids (VFA) was unaffected (P > 0.05) by EFE application, doses, and application methods in MS and OS. However, total and individual VFA increased (P < 0.05) when SCB was incubated with xylanase and this was EZ dose dependent (P < 0.05). Methane and CO₂ concentrations were not affected (P > 0.05) with EZ application methods or its doses in MS and OS. However, increasing EFE doses with SCB increased CH₄ (P = 0.013) and CO₂ concentrations (P = 0.006). It could be concluded that application of xylanase enzyme improved GP and rumen fermentation activities. However, the effects were substrate, application methods, and EFE doses dependent, and increasing EZ doses had more effects than low doses.