The present work aims to study the effect of novel sub-transus heat treatment on the microstructure evolution and hardness of Ti6Al4V alloy fabricated using laser based powder bed fusion. The results revealed that the as-built microstructure is α′ martensite which decomposes to α + β during sub-transus heat treatment at 970 °C for different soaking times. The formation of a bi-modal structure with approximately 25% globular primary α and transformed β is observed. The development of globular primary alpha is attributed to thermal grooving and boundary splitting. The resultant coarsening of these α/β grains was associated with a time-temperature combination and orientation relation (OR) to 45° [100]. Moreover, precipitation of fine Ti₃Al intermetallic (α₂) in plate form is observed predominantly inside the primary α grains. Owing to the faster cooling rate post soaking time resulted in Ti₃Al precipitates which is the conversion of α to α₂ precipitates. The average micro-hardness of the specimen heat-treated for 12 h at 970 °C is found to be 45% higher in comparison to the as-built specimen. Whereas, the micro-hardness differs between the primary α and transformed β region due to the alloy element partitioning effect. The average micro-hardness of 592 HV is observed in primary α grains in the specimen heat-treated for 12 h at 970 °C. The prior results are associated with the formation of intermetallic nano-sized Ti₃Al (α₂).