Aims: Oil palm trunk (OPT) can be a potential biomass from replanting activities for biomass-to-liquid (BTL) particularly in bioethanol production. The OPT contains higher carbohydrates compared to other oil palm biomass, thus has better advantages as feedstock for biofuel. To realise this, the feasibility of using oil palm trunk (OPT) sap as a substrate for bioethanol fermentation was explored via optimising the various culture conditions (pH, temperature, inoculum size, nitrogen source, dilution effect and growth medium) using Saccharomyces cerevisiae. Methodology and results: A total of six parameters were tested for optimising bioethanol production i.e. pH, temperature, inoculum size, nitrogen source, dilution effect and types of medium. Results showed that the optimum conditions for OPT sap in bioethanol production were at pH 4.0, temperature of 30 °C, inoculum size of 10 % (v/v), without requirement of nitrogen supplementation and substrate dilution. A fermentation period of 24 h was best for bioethanol production and resulted in bioethanol production, formation rate and yield of 47.5 g/L, 1.98 g/h and 0.50 g/g, respectively. Conclusion, significance and impact study:The study has clearly demonstrated that high efficient bioethanol production from OPT sap is possible but it is susceptible to various fermentation influencing parameters. This study could establish an effective and sustainable utilisation of waste OPT especially its sap as a lignocellulosic biomass supplement from the oil palm industry for second generation biofuel production.