Experimental and chemical kinetics studies were carried out to compare soot particle morphology evolution in atmospheric pressure laminar co-flow diffusion flames of methyl butanoate (MB) and ethyl butanoate (EB), in order to investigate the effects of alcohol chain in biodiesel molecular on soot formation. The thermophoretic sampling technique was used to capture soot particles directly at different heights along flame centerlines. Transmission electron microscopy (TEM) was applied to obtain particle morphology information. Moreover, total sampling followed by thermogravimetric analysis and Raman spectroscopy analysis was performed to study soot oxidation reactivity and degree of disorder in soot structure. The results show that the flame structures and temperature profiles along flame centerlines of two test esters are similar. The primary particle diameters of EB are larger at almost all the sampling positions, and soot inception and aggregation process occur earlier in EB flame. The phenomenon can be illustrated through simulation work which indicated that EB could be decomposed quickly at lower temperature because of the six-centered unimolecular elimination reaction, leading to earlier and stronger formation of C2H4, and then promoting the formation of C2H2. It is also found that the oxidation reactivity of soot from MB flame becomes higher compared with that of EB.