For the cold pre-deformed Ti-10Mo-8V-1Fe-3.5Al (TB3) alloy, the high density of dislocation could still be observed after traditional aging treatment, which could lead to detrimental influences on mechanical properties. To resolve this problem, a second aging treatment process was proposed. Transmitting electron microscopy (TEM) investigations show that the dislocation density may be dramatically decreased by the secondary aging treatment in predeformed TB3 samples. Variant selection effect was observed in predeformed and aged TB3 samples. Low cycle fatigue (LCF) tests were carried out to the conventional peak aged TB3 samples (FA) and the predeformed and secondary aging treated TB3 samples(SA). The LCF results show that fatigue lifetime is slightly higher for SA samples when the total strain amplitude is lower than 1%, but it has an opposite result when the total strain amplitude is higher than 1%. TEM investigations on the TB3 samples after fatigue tests show that dislocation slip is the predominant deformation mode. The dislocation bands resulting form cyclic deformation are wide and discontinuous in FA sample, however they are fine and homogeneous in SA samples.