Di-n-butyl ether (DBE), a promising lignocellulosic biofuel, has been suggested as a potential alternative fuel for compression ignition engines. In this study, the spray auto-ignition characteristics of diesel/DBE blends were experimentally measured on a constant volume combustion chamber. Time-resolved pressure traces and heat release rates in fuel spray combustion were measured at changed fuel blending fractions, ambient temperatures, and oxygen concentrations. Further, ignition delay and combustion delay that evaluates fuel spray ignition tendency were derived and compared for different test blends. Experimental results indicated that fuel spray ignition tendency is promoted with DBE addition, evidenced by the advanced pressure rise and heat release processes, and the shortened ignition and combustion delays. Peak heat release rates are fuel-dependent at high ambient oxygen concentrations since the relative fractions of the premixed and diffusive burns alter with changed DBE addition. However, as the oxygen concentration drops to 11%, fuel effects on the peak heat release rates become less noticeable. Reduced ambient oxygen concentration effectively extends fuel ignition and combustion delays, and typical two-stage pressure rises and heat releases are observed for all test blends, as the oxygen concentration drops to 11%.