Effects of three different cell pretreatment methods on microalgae Isochrysis sp. pyrolysis kinetics, biocrude yields and quality, energy conversion efficiency and life cycle assessment (LCA) were investigated. From derived thermogrametry (DTG) curves, the decomposition reaction was apparently enhanced for acid pretreatment sample at 200°C. The activation energy of pretreated microalgae pyrolysis was lower than that without pretreatment. The biocrude yields for raw microalgae were higher than pretreated samples at 450–475°C and lower at 400–425°C. Carbon distributions of biocrude from microalgae pyrolysis with ultrasonication and microwave pretreatment were similar and mainly in C6-10, C16, C18, and C20, while carbon distribution of biocrude from acid pretreatment mainly located in C7, C16, C18, and C20. Acid heating pretreatment improved fuel quality including N compounds reduction and increase of esters and ethers content, which was likely due to esterification reaction between carbohydrate derivative and lipid at acid conditions. From the point of energy conversion efficiency, pretreatment decreased the energy ratio and energy efficiency and increased the energy consumption/output ratio ECR (except ECR of acid heating pretreatment) during microalgae pyrolysis process. Based on the LCA, pretreatment increased the GHG emissions in the production process of alternative biofuels. Co-use of pretreatment process during biofuel production and valuable chemicals extraction can be further researched to enhance energy efficiency and cost-efficiency for microalgae.