Chlorella is a rich source of nutrients. In addition to its nutritional value, it exhibits versatile biological activities. New strains have been extensively identified and investigated in recent years to expand the potential of Chlorella. The accurate measurement of pulse-amplitude-modulated (PAM) fluorometry parameters and effective microalgal cell lysis are foundational for advanced studies of novel Chlorella species. In this study, ribosomal small subunit (SSU)-internal transcribed spacer (ITS) phylogenetic analysis and internal transcribed spacer 2 (ITS2) secondary structure analysis were employed to identify a new Chlorella species. The dark adaptation time, the duration of the saturation pulse, the intensity of actinic light, and the duration of actinic light exposure for PAM fluorometry measurements were optimized. Different conditions of ultrasonication and high-pressure homogenization (HPH) for microalgal cell lysis were compared. Chlorella vulgaris K-01 was identified. The suitable duration for dark adaptation, the saturation pulse, and the actinic light were 15 min, 200 milliseconds, and 30 s, respectively. The suitable intensity of actinic light was 191 μE/(m(2)·s). For microalgal cell lysis, HPH could achieve 98.65% cell lysis efficiency at 30 kpsi (207 MPa), whereas ultrasonication attained an efficiency of 45.47% (300 W for 30 min). These results facilitate further study on the physiology and the composition of Chlorella vulgaris K-01.