Given their wide range of biomedical applications, hydroxyapatite (HA) nanoparticles are an attractive material widely used in many fields. Therefore, a simple, inexpensive, and stable process for the synthesis of HA nanoparticles is necessary to meet current needs. Herein, we studied HA synthesis assisted by four surfactants, namely cation, anion, non-ionic, and zwitterion templates, to verify the synthesis phase, aspect ratio, morphology, and biocompatibility under different environments (i.e., pH 4 and 9) before and after calcination. Results showed that before calcination, the surfactant-free groups could not produce HA but showed an abundant dicalcium phosphate anhydrous (DCPA) phase at pH 4. Except for the anionic group containing a small amount of DCPA, all surfactant-assistant groups presented single-phase HA in acidic and alkaline environments. The diameter of HA synthesized at pH 4 was significantly larger than that of HA synthesized at pH 9, and the effect of aspect ratio changes after calcination was more significant than that before calcination. The uncalcined rod-shaped HA synthesized with a non-ionic template at pH 4 demonstrated excellent cell viability, whereas anionic, cationic, and non-ionic surfactants exhibited biocompatibility only after calcination. At pH 9, non-ionic and uncalcined zwitterion-assisted rod-shaped HA showed excellent biocompatibility. In conclusion, the uncalcined HA rod-shaped nanoparticles synthesized from the non-ionic template at pH 4 and 9 and the zwitterion template at pH 9, as well as all surfactant-assisted HA after calcination, had no cytotoxicity. These tailor-made non-toxic HA types can meet the different requirements of apatite composite materials in biomedical applications.