Micropropagation protocols for red-fleshed Hylocereus species (Cactaceae) have been developed; however, these methods prolong the sprout duration from areoles and produce irregular micro-propagules in ‘Da Hong’ pitaya. Thus, the present study aimed to establish an improved micropropagation protocol for this cultivar. Shoot regeneration and root induction of self-pollinating seedling segments were evaluated in response to combinations of activated charcoal (AC; 200 mg/L), α-naphthaleneacetic acid (NAA; 0.05, 0.10, and 0.20 mg/L), and 6-benzylaminopurine (BAP; 1.00, 2.00, and 4.00 mg/L). The correlations among plantlet growth characteristics and plantlet survival rate after transplantation under field conditions were calculated. Increasing the NAA concentration increased the number of roots but reduced root length. The addition of AC enhanced shoot length and prevented the regeneration of dried-out, clustered, and abnormal shoots. Plantlets treated with 200 mg/L AC and 0.10 mg/L NAA produced the highest number of shoots, i.e., 4.1 shoots, which however, were shorter and lighter than those cultured with AC alone. Plantlets grown on medium supplemented with BAP showed no advantage in shoot number, shoot weight, plantlet surface area, or plantlet volume. The weight and shoot surface area of plantlets were strongly correlated. All plantlets grew well at 4 weeks post-transplantation. Overall, these results support this improved micropropagation method to regenerate robust ex vitro plantlets.