DNA origami nanocarriers have emerged as a promising tool for many biomedical applications, such as biosensing, targeted drug delivery, and cancer immunotherapy. These highly programmable nanoarchitectures are assembled into any shape or size with nanoscale precision by folding a single-stranded DNA scaffold with short complementary oligonucleotides. The standard scaffold strand used to fold DNA origami nanocarriers is usually the M13mp18 bacteriophage’s circular single-stranded DNA genome with limited design flexibility in terms of the sequence and size of the final objects. However, with the recent progress in automated DNA origami design—allowing for increasing structural complexity—and the growing number of applications, the need for scalable methods to produce custom scaffolds has become crucial to overcome the limitations of traditional methods for scaffold production. Improved scaffold synthesis strategies will help to broaden the use of DNA origami for more biomedical applications. To this end, several techniques have been developed in recent years for the scalable synthesis of single stranded DNA scaffolds with custom lengths and sequences. This review focuses on these methods and the progress that has been made to address the challenges confronting custom scaffold production for large-scale DNA origami assembly.