The Night Shift features Dr. Drew Alister, a central gay character whose personal and romantic life is a significant narrative thread. His relationship with Rick is depicted with dignity, complexity, and respect, showcasing their commitment, marriage, and desire to build a family. The series consistently affirms the worth of LGBTQ+ lives and love through Drew's positive and nuanced portrayal.

The Night Shift features a transgender character whose identity is portrayed positively through the affirming actions of the medical staff. The narrative addresses external challenges like parental non-acceptance and medical issues, consistently validating the character's identity and advocating for respectful, empathetic care.

The series frequently depicts patients and their families drawing strength and comfort from their Christian faith during medical crises, treating these beliefs with respect and empathy. While characters may face existential challenges, the narrative consistently frames individual faith as a valid and often supportive aspect of coping.